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THIRTEEN
CHAPTER 13
Probes for Lipids and Membranes
13.1 Introduction to Membrane Probes .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 547
Fluorescent and Biotinylated Membrane Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 547
Fluorescent Analogs of Natural Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 547
Other Lipophilic and Amphiphilic Fluorescent Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 548
Other Probes for Studying Cell Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 548
13.2 Fatty Acid Analogs and Phospholipids
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 549
Fluorescent Fatty Acid Analogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 549
BODIPY® Fatty Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 549
NBD Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 551
Pyrene Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 551
Dansyl Undecanoic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 551
cis-Parinaric Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 551
ADIFAB Fatty Acid Indicator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 553
Phospholipids with BODIPY® Dye–Labeled Acyl Chains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 554
BODIPY® Glycerophospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 554
Applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 556
Phospholipid with DPH-Labeled Acyl Chain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557
Properties. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557
Applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557
Phospholipids with NBD-Labeled Acyl Chains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557
Properties. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557
Applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557
Phospholipids with Pyrene-Labeled Acyl Chains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 558
Properties. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 558
Applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 558
Phospholipids with a Fluorescent or Biotinylated Head Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559
Phospholipid with a Dansyl-Labeled Head Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559
Phospholipid with a Marina Blue® Dye–Labeled Head Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559
Phospholipid with a Pacific Blue™ Dye–Labeled Head Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559
Phospholipid with an NBD-Labeled Head Group. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559
Phospholipid with a Fluorescein-Labeled Head Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559
Phospholipid with an Oregon Green® 488 Dye–Labeled Head Group. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560
Phospholipid with a BODIPY® FL Dye–Labeled Head Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560
Phospholipids with a Rhodamine or Texas Red® Dye–Labeled Head Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560
Phospholipids with a Biotinylated Head Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560
LipidTOX™ Phospholipid and Neutral Lipid Stains for High-Content Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561
HCS LipidTOX™ Phospholipidosis Detection Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561
HCS LipidTOX™ Neutral Lipid Stains. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561
HCS LipidTOX™ Phospholipidosis and Steatosis Detection Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 562
Data Table 13.2 Fatty Acid Analogs and Phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563
Product List 13.2 Fatty Acid Analogs and Phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 565
13.3 Sphingolipids, Steroids, Lipopolysaccharides and Related Probes
. . . . . . . . . . . . . . . . . . . . 566
Sphingolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 566
Structure and Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 566
BODIPY® Sphingolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 567
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Chapter 13 — Probes for Lipids and Membranes
NBD Sphingolipids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 568
Vybrant® Lipid Raft Labeling Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 568
Amplex® Red Sphingomyelinase Assay Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 569
Steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 569
BODIPY® Cholesteryl Esters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 569
Side Chain–Modified Cholesterol Analog . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 570
Amplex® Red Cholesterol Assay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 570
Fluorescent Triacylglycerol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571
Lipopolysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571
Fluorescent Lipopolysaccharides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571
Pro-Q® Emerald 300 Lipopolysaccharide Gel Stain Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572
Data Table 13.3 Sphingolipids, Steroids, Lipopolysaccharides and Related Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 573
Product List 13.3 Sphingolipids, Steroids, Lipopolysaccharides and Related Probes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574
13.4 Dialkylcarbocyanine and Dialkylaminostyryl Probes
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575
Dialkylcarbocyanine Probes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575
DiI, DiO, DiD, DiR and Analogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575
Spectral Properties of Dialkylcarbocyanines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575
Substituted DiI and DiO Derivatives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 576
DiI and DiO as Probes of Membrane Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577
DiI and DiO as Probes of Membrane Dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577
Dialkylaminostyryl Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577
Data Table 13.4 Dialkylcarbocyanine and Dialkylaminostyryl Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 578
Product List 13.4 Dialkylcarbocyanine and Dialkylaminostyryl Probes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579
13.5 Other Nonpolar and Amphiphilic Probes
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579
Amphiphilic Rhodamine, Fluorescein and Coumarin Derivatives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579
Octadecyl Rhodamine B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579
Amphiphilic Fluoresceins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580
Amphiphilic Coumarin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580
DPH and DPH Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580
Diphenylhexatriene (DPH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580
TMA-DPH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581
Nonpolar BODIPY® Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581
BODIPY® Fluorophores. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581
BODIPY® FL C5-Ceramide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 582
CellTrace™ BODIPY® TR Methyl Ester . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 582
Pyrene, Nile Red and Bimane Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 582
Nonpolar Pyrene Probe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 582
Nile Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 582
Bimane Azide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 583
LipidTOX™ Neutral Lipid Stains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 583
Membrane Probes with Environment-Sensitive Spectral Shifts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584
Prodan and Laurdan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584
Dapoxyl® Derivative . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584
Anilinonaphthalenesulfonate (ANS) and Related Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584
Bis-ANS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585
DCVJ. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585
Data Table 13.5 Other Nonpolar and Amphiphilic Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 586
Product List 13.5 Other Nonpolar and Amphiphilic Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587
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Chapter 13 — Probes for Lipids and Membranes
Section 13.1 Introduction to Membrane Probes
13.1 Introduction to Membrane Probes
Fluorescent and Biotinylated Membrane Probes
The plasma membranes and intracellular membranes of live cells and the artificial membranes of liposomes represent a significant area of application for fluorescent probes. Membrane
probes include fluorescent analogs of natural lipids, as well as lipophilic organic dyes that have
little structural resemblance to natural biomolecules. We offer a wide range of both types of
membrane probes. These probes are used for structural and biophysical analysis of membranes,
for following lipid transport and metabolism in live cells (Figure 13.1.1) and for investigating
synaptosome recycling (Section 16.1) and lipid-mediated signal transduction processes (Chapter
17). Due to their low toxicity and stable retention, some lipid probes are particularly useful for
long-term cell tracing (Section 14.4). Other, slightly less lipophilic probes are used as membrane
markers of endocytosis and exocytosis (Section 16.1).
Fluorescent Analogs of Natural Lipids
We offer fluorescent and, in a few cases, biotinylated analogs of five naturally occurring lipid
classes—phospholipids, sphingolipids (including ceramides), fatty acids, triglycerides and steroids. Phospholipids are the principal building blocks of cell membranes. Most phospholipids are
derivatives of glycerol comprising two fatty acyl residues (nonpolar tails) and a single phosphate
ester substituent (polar head group). Despite their overall structural similarity (Figure 13.1.2),
natural phospholipids exhibit subtle differences in their fatty acid compositions, degree of acyl
A
HOCH
+
(CH ) NCH CH O
33
2
2
O
CH
CH
OH
OH
2
Glycerol
O
P
2
Figure 13.1.1 The cytoplasm of a live zebrafish embryo
labeled with the green-fluorescent lipophilic tracer BODIPY®
505/515 (D3921). The image was contributed by Arantza
Barrios, University College, London.
-
O CH
CH
CH
O
O
O C
C
R
R
2
2
O
O
P
O
-
O CH
O
Phosphatidylcholine
O
OH
O P O CH
2
O
OH
OH
HO
OH
CH
CH
O
O
O C
C
R
R
CH
CH
O
O
O C
C
R
R
2
2
O
Phosphatidic Acid
2
O
Phosphatidylinositol
B
HOCH
2
CH
NH
+
(CH ) NCH CH O
33
2 2
CH
2
O
CH(CH ) CH
2 12 3
Sphingosine
O
P
CH
OH
-
O CH
2
CH
CH
NH
OH
C
CH
HO
CH(CH ) CH
2 12
3
OH
O
HO
R
Sphingomyelin
O O CH 2
2
CH
CH
NH
OH
C
CH
CH(CH ) CH
2 12
3
CH
NH
OH
C
OH
HOCH
CH
CH
CH(CH ) CH
2 12
3
O
R
Cerebroside
O
R
Ceramide
Figure 13.1.2 A) Phosphatidylcholines, phosphatidylinositols and phosphatidic acids are examples of glycerolipids derived from glycerol. B) Sphingomyelins, ceramides
and cerebrosides are examples of sphingolipids derived from sphingosine. In all the structures shown, R represents the hydrocarbon tail portion of a fatty acid residue.
™
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TheMolecular
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Handbook:
A Guide
to Fluorescent
Probes
and
Labeling
Technologies
IMPORTANT
NOTICE:described
The products
described
thiscovered
manual are
by oneLimited
or moreUse
Limited
Use
Label License(s).
to the
Appendix
IMPORTANT NOTICE
: The products
in this
manualinare
by covered
one or more
Label
License(s).
PleasePlease
referrefer
to the
Appendix
on on
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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547
Chapter 13 — Probes for Lipids and Membranes
Section 13.1 Introduction to Membrane Probes
chain unsaturation and type of polar head group.1 These differences can
produce significant variations in membrane physical properties, in the
location of phospholipids in a lipid bilayer and in their biological activity. Fluorescent phospholipid analogs (Section 13.2) can be classified
according to where the fluorophore is attached. The fluorophore can be
attached to one (or both) of the fatty acyl chains or to the polar head
group. The attachment position of the fluorophore determines whether
it is located in the nonpolar interior or at the water/lipid interface when
the phospholipid analog is incorporated into a lipid bilayer membrane.
Fatty acids are the building blocks for a diverse set of biomolecules. Some fatty acids (e.g., arachidonic acid) are important in cell
signaling.2 Fatty acids are liberated by the enzymatic action of phospholipase A on phospholipids (Section 17.4) and also by various other lipases. Fluorescent fatty acids can often be used interchangeably with the
corresponding phospholipids as membrane probes; however, fatty acids
transfer more readily between aqueous and lipid phases.3 Although fatty acids are ionized at neutral pH in water (pKa ~5), their pKa is typically
~7 in membranes, and thus a significant fraction of membrane-bound
fatty acids are neutral species.3 Certain fluorescent fatty acids (Section
13.2) are readily metabolized by live cells to phospholipids, mono-, diand triacylglycerols, cholesteryl esters and other lipid derivatives.4
Sphingolipids play critical roles in processes such as proliferation,
apoptosis, signal transduction and molecular recognition at cell surfaces.1,5,6 Defects in the lysosomal breakdown of sphingolipids are the
underlying cause of lipid storage disorders such as Niemann–Pick, Tay–
Sachs, Krabbe and Gaucher diseases. The sphingolipids described in
Section 13.3 include ceramides, sphingomyelins, glycosylceramides (cerebrosides) and gangliosides. The structural backbone of sphingolipids
is the lipophilic amino–dialcohol sphingosine (2-amino-4-octadecen1,3-diol, Figure 13.1.2) to which a single fatty acid residue is attached via
an amide linkage. Our fluorescent analogs of sphingolipids are prepared
by replacing the natural amide-linked fatty acid with a fluorescent analog. Sphingolipids with an unmodified hydroxyl group in the 1-position
are classified as ceramides. As part of the lipid-sorting process in cells,
ceramides are glycosylated to cerebrosides (Figure 13.1.2) or converted
to sphingomyelins (Figure 13.1.2) in the Golgi complex. Glycosylated
sphingolipids (cerebrosides and gangliosides) occur in the plasma membranes of all eukaryotic cells and are involved in cell recognition, signal
transduction and modulation of receptor function.7 Gangliosides have
complex oligosaccharide head groups containing at least one sialic acid
residue in place of the single galactose or glucose residues of cerebrosides.
Fluorescent cholesteryl esters and triglycerides (Section 13.3) can
be used as structural probes and transport markers for these important
lipid constituents of membranes and lipoproteins.8 They may also serve
as fluorescent substrates for lipases and lipid-transfer proteins and can
be incorporated into low-density lipoproteins (LDL, Section 16.1).
Other Lipophilic and Amphiphilic Fluorescent Probes
The probes described in Section 13.4 and Section 13.5 are not analogs of any particular biological lipid class, but they have a general structural resemblance that facilitates labeling of membranes, lipoproteins
and other lipid-based molecular assemblies. Particularly notable members of this group are the lipophilic carbocyanines DiI (Figure 13.1.3),
DiO, DiD and DiR, the lipid fluidity probes DPH and TMA-DPH and
the membrane-surface probes ANS and laurdan. These probes generally
have limited water solubility and exhibit substantially enhanced fluorescence upon partition into lipid environments. They can be classified
as either amphiphilic (having both polar and nonpolar structural elements) or neutral (lacking charges and most soluble in very nonpolar
environments). We use similar neutral lipophilic dyes for internal staining of our fluorescent polystyrene microspheres (Section 6.5).
Other Probes for Studying Cell Membranes
In addition to the lipophilic probes described in this chapter, we
have available the following products for studying the properties and
functions of cell membranes:
• Moderately lipophilic stains for the endoplasmic reticulum and
Golgi apparatus (Section 12.4)
• FM® dyes—amphiphilic probes for cell membrane labeling (Section
14.4, Section 16.1)
• CellLight® Plasma Membrane-CFP, CellLight® Plasma MembraneGFP and CellLight® Plasma Membrane-RFP, which are BacMam
2.0 vectors encoding fluorescent proteins targeted to the plasma
membrane (C10606, C10607, C10608; Section 14.4)
• Alexa Fluor® dye–labeled cholera toxin subunit B conjugates for
labeling lipid rafts (Section 14.7)
• Annexin V conjugates for detection of phosphatidylserine exposure in apoptotic cell membranes (Section 15.5)
• Fluorescent and fluorogenic phospholipase A substrates (Section 17.4)
• Amplex® Red Phosphatidylcholine-Specific Phospholipase C Assay
Kit and Amplex® Red Phospholipase D Assay Kit (A12218, A12219;
Section 17.4)
• Antibodies to phosphatidylinositol phosphates (Section 17.4)
• Lipophilic pH indicators (Section 20.4)
• Membrane potential–sensitive probes (Section 22.2, Section 22.3)
REFERENCES
Figure 13.1.3 The neuronal tracer DiI (D282, D3911) used as a diagnostic tool to evaluate
patterns of innervation in newborn mouse cochlea. The larger image is of a mutant cochlea
and the inset is of a wild-type cochlea. Image contributed by Bernd Fritzsch, Creighton
University, and L. Reichardt and I. Farinas, Howard Hughes Medical Institute, San Francisco.
1. Nat Rev Mol Cell Biol (2008) 9:112; 2. Biochim Biophys Acta (1994) 1212:26; 3. J Lipid
Res (1998) 39:467; 4. Chem Phys Lipids (1991) 58:111; 5. Annu Rev Biochem (1998)
67:27; 6. Biochim Biophys Acta (1991) 1082:113; 7. Ann N Y Acad Sci (1998) 845:57;
8. Nat Rev Mol Cell Biol (2008) 9:125.
The
MolecularProbes®
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Probes and
and Labeling
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The
Molecular
Guideto
toFluorescent
Fluorescent Probes
™
548
IMPORTANT
NOTICE:
The products
described
in this manual
coveredare
by covered
one or more
Limited
Use Label
License(s).
Please
refer to thePlease
Appendix
onto
IMPORTANT
NOTICE
: The products
described
in thisaremanual
by one
or more
Limited
Use Label
License(s).
refer
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
the Appendix on
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
www.invitrogen.com/probes
thermofisher.com/probes
Chapter 13 — Probes for Lipids and Membranes
Section 13.2 Fatty Acid Analogs and Phospholipids
13.2 Fatty Acid Analogs and Phospholipids
The probes in this section and in Section 13.3 bear some structural
resemblance to natural lipids. Included in this section are fluorescent
fatty acid analogs, as well as phospholipids wherein one or both fatty
acid esters are replaced by fluorescent fatty acid esters. The fluorophores
in these probes tend to remain buried in the hydrophobic interior of
lipid bilayer membranes.1,2 In this location, they are sensitive to membrane properties such as lipid fluidity, lateral domain formation and
structural perturbation by proteins, drugs and other additives. Also
included in this section are several head group–modified phospholipid
analogs incorporating a fluorophore or biotin (Table 13.1).
Sphingolipids, steroids and lipopolysaccharides are discussed
in Section 13.3. Important applications of the fluorescent phosphatidylinositol derivatives as probes for signal transduction and various
fluorescent phospholipids as phospholipase substrates are further described in Section 17.4. A review of fluorescent lipid probes and their
use in biological and biophysical research has been published.3
Fluorescent Fatty Acid Analogs
Our fluorescent fatty acid analogs have a fluorophore linked within
the fatty acid chain or, more commonly, at the terminal (omega) carbon
atom that is furthest from the carboxylate moiety. Although fluorescent
fatty acid analogs are sometimes used as direct probes for membranes
and liposomes, their most common applications have been for synthesis of fluorescent phospholipids and for metabolic incorporation by live
cells. Our fluorescent fatty acids currently include derivatives based on
the BODIPY®, nitrobenzoxadiazole (NBD), pyrene and dansyl fluorophores, as well as the naturally fluorescent polyunsaturated fatty acid,
cis-parinaric acid.
BODIPY® Fatty Acids
BODIPY® fatty acids are, by far, the most fluorescent fatty acid analogs that we have available.4 The lack of ionic charge on the BODIPY®
fluorophore is unusual for long-wavelength fluorescent dyes and results in exclusive localization of the fluorophore within the membrane
(Figure 13.2.1). BODIPY® derivatives typically have extinction coefficients greater than 90,000 cm–1M–1 with absorption maxima beyond
500 nm. A useful spectroscopic property of BODIPY® dyes is the concentration-dependent formation of excited-state dimers ("excimers")
with red-shifted emission. We have observed this phenomenon particularly with our green-fluorescent BODIPY® fatty acid derivatives,
which undergo a considerable red shift in their emission when metabolically incorporated into lipophilic products 5 (Figure 13.2.2). Pyrene
A
E
O
C
O
-
O
N
O
2
N
N
NH
O
B
C
O
F
O
F
N
B
F
C
O
_
N
G
O
Figure 13.2.2 BHK cells incubated in medium containing the fluorescent fatty acid analog
BODIPY® 500/510 C1,C12 (D3823). This photomicrograph, obtained through a standard fluorescein longpass filter set, reveals reticular green-fluorescent staining as well as yelloworange–fluorescent spherical structures. These fluorescent structures are indicative of
the metabolic accumulation of BODIPY® dye–labeled neutral lipids in cytoplasmic droplets. Image contributed by Juha Kasurinen, University of Helsinki, Finland.
O
+
N( CH CH )
2
3 2
O
( CH CH ) N
3
2 2
C
C
O
SO
3
C
_
O
SO
2
NH
Table 13.1 Phospholipids with labeled head groups.
Label (Ex/Em) *
Dansyl (336/517)
Marina Blue® (365/460)
Pacific Blue™ (410/455)
NBD (463/536)
Fluorescein (496/519)
Oregon Green® 488 (501/526)
BODIPY® FL (505/511)
BODIPY® 530/550
Tetramethylrhodamine (540/566)
Lissamine rhodamine (560/581)
Texas Red® (582/601)
Biotin (<250/none)
Cat. No.
D57
M12652
P22652
N360
F362
O12650
D3800
D3815
T1391
L1392
T1395MP
B1550, B1616
* Fluorescence excitation (Ex) and emission (Em) spectral maxima, in nm, are in
methanol. The spectra may be different in membranes.
H
D
H C
3
N
+
N CH
3
N
H C
3
CH
CH
_
H C
3
O
O
CH
H C
3
N
O
+
_
C
O
O
C
I
NH
O
Figure 13.2.1 Location and orientation of representative fluorescent membrane probes in a
phospholipid bilayer: A) DPH (D202), B) NBD-C6-HPC (N3786), C) bis-pyrene-PC (B3782), D) DiI
(D282), E) cis-parinaric acid (P36005), F) BODIPY® 500/510 C4, C9 (B3824), G) N-Rh-PE (L1392),
H) DiA (D3883) and I) C12-fluorescein (D109).
™
The
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A Guide
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and
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TheMolecular
MolecularProbes
Probes®
Handbook:
A Guide
to Fluorescent
Probes
and
LabelingTechnologies
Technologies
IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on
IMPORTANT NOTICE
: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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549
Chapter 13 — Probes for Lipids and Membranes
Section 13.2 Fatty Acid Analogs and Phospholipids
H3C
N
H3 C
F
B
N
O
F
(CH2)15
C OH
Figure 13.2.3 4,4-Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid
(BODIPY® FL C16, D3821).
O
N
F
B
N
C
F
OH
C -BODIPY® 500/510 C12
1
N
F
B
N
O
F
C
OH
C -BODIPY® 500/510 C9
4
N
F
B
N
O
F
C
OH
C -BODIPY® 500/510 C5
8
Figure 13.2.4 Structural representations showing the positional shift of the fluorophore
with respect to the terminal carboxyl group in a homologous series of BODIPY® 500/510 fatty
acids (D3823, B3824, D3825).
N
F
B
N
O
F
(CH2)11
C OH
Figure 13.2.5 4,4-Difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid
(BODIPY® 530/550 C12, D3832).
N
F
B
N
fatty acids also exhibit excimer formation but their emission is at much
shorter wavelengths than that of the BODIPY® dyes and they are therefore less suitable for the study of live cells.
The fluorophores in our current selection of BODIPY® fatty acids
and their approximate absorption/emission maxima (in nm) are:
• BODIPY® 503/512 (BODIPY® FL; D3821, Figure 13.2.3; D3822;
D3834; D3862)
• BODIPY® 500/510 (D3823, B3824, D3825; Figure 13.2.4)
• BODIPY® 530/550 (D3832, Figure 13.2.5)
• BODIPY® 558/568 (D3835, Figure 13.2.6)
• BODIPY® 581/591 (D3861)
BODIPY® fatty acids are synthetic precursors to a wide variety of
fluorescent phospholipids (described below), as well as several important sphingolipid probes described in Section 13.3. Some BODIPY® fatty acids are readily metabolized by live cells to phospholipids, di- and
triacylglycerols, cholesteryl esters and other natural lipids.6–9 Analysis
of cellular lipid extracts by HPLC has shown that glycerophosphocholines constitute more than 90% of the products of biosynthetic incorporation of BODIPY® 500/510 dodecanoic acid (D3823) by BHK cells.5
The three BODIPY® 500/510 probes form a unique series in which
the green-fluorescent fluorophore is located within the fatty acid chain
at different distances from the terminal carboxylate group.4 The overall
length of the probe is constant and, including the fluorophore, is about
equivalent to that of an 18-carbon fatty acid (Figure 13.2.4).
BODIPY® 581/591 undecanoic acid (D3861) is particularly useful for detecting reactive oxygen species in cells and membranes.10–13
Oxidation of the polyunsaturated butadienyl portion of the BODIPY®
581/591 dye (Figure 13.2.7) truncates the conjugated π-electron system,
resulting in a shift of the fluorescence emission peak from ~590 nm to
~510 nm.10,13,14 This oxidation response mechanism is similar to that of
the naturally occurring polyunsaturated fatty acid cis-parinaric acid. In
comparison to cis-parinaric acid, advantages of BODIPY® 581/591 undecanoic acid include:
• Long-wavelength excitation, compatible with confocal laser-scanning microscopes and flow cytometers
• Avoidance of photooxidation effects induced by ultraviolet
excitation
• Less interference by colored oxidant and antioxidant additives
when detecting probe fluorescence 12
• Greater resistance to spontaneous oxidation
• Red-to-green fluorescence shift, allowing the use of fluorescence
ratio detection methods 10,13
O
F
(CH2)11
�
C OH
Figure 13.2.6 4,4-Difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid
(BODIPY® 558/568 C12, D3835).
Figure 13.2.7 4,4-Difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3undecanoic acid (BODIPY® 581/591 C11, D3861).
TheMolecular
MolecularProbes®
Probes Handbook:
Handbook: AAGuide
and Labeling
LabelingTechnologies
Technologies
The
Guideto
toFluorescent
Fluorescent Probes
Probes and
™
550
IMPORTANT
NOTICE:
The products
described
in this manual
covered
bycovered
one or more
Limited
Use Label
License(s).
Please
refer to thePlease
Appendix
IMPORTANT
NOTICE
: The products
described
in thisare
manual
are
by one
or more
Limited
Use Label
License(s).
referonto
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
the Appendix on
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
www.invitrogen.com/probes
thermofisher.com/probes
Chapter 13 — Probes for Lipids and Membranes
Section 13.2 Fatty Acid Analogs and Phospholipids
O
An alternative technique for detecting lipid peroxidation utilizes the oxidation-induced decrease of concentration-dependent excimer formation by BODIPY® FL dye–labeled fatty acids.15
NH(CH2)5
N
NBD Fatty Acids
Fluorescence of the nitrobenzoxadiazole (NBD) fluorophore is highly sensitive to its environment. Although it is moderately fluorescent in aprotic solvents, in aqueous solvents it is almost nonfluorescent.16 The NBD fluorophore is moderately polar and both its homologous 6-carbon and 12-carbon fatty acid analogs (N316, Figure 13.2.8; N678) and the phospholipids derived
from these probes (N3786, N3787) tend to sense the lipid–water interface region of membranes
instead of the hydrophobic interior 17 (see part B of Figure 13.2.1). The environmental sensitivity
of NBD fatty acids can be usefully exploited to probe the ligand-binding sites of fatty acid and
sterol carrier proteins.18 NBD fatty acids are not well metabolized by live cells.9,19
N
C OH
O
NO2
Figure 13.2.8 NBD-X (6-(N-(7-nitrobenz-2-oxa-1,3-diazol4-yl)amino)hexanoic acid; N316).
Pyrene Fatty Acids
Figure 13.2.9 1-Pyrenedodecanoic acid (P96).
Fluorescence emission
The hydrophobic pyrene fluorophore is readily accommodated within the membrane.20
ω-Pyrene derivatives of longer-chain fatty acids (Figure 13.2.9) were first described by Galla and
Sackmann in 1975.21 We offer pyrene derivatives of the 4-, 10-, 12- and 16-carbon fatty acids
(P1903MP, P31, P96, P243, respectively). Pyrenebutanoic acid—frequently called pyrenebutyric
acid (P1903MP)—has rarely been used as a membrane probe; however, its conjugates have exceptionally long excited-state lifetimes (τ >100 nanoseconds) and are consequently useful for
time-resolved fluorescence immunoassays and nucleic acid detection.22,23 The long excited-state
lifetime of pyrenebutyric acid also makes it useful as a probe for oxygen in cells 24–27 and lipid
vesicles.28
Pyrene derivatives form excited-state dimers (excimers) with red-shifted fluorescence emission 29–31 (Figure 13.2.10). Pyrene excimers can even form when two pyrenes are tethered by a
short trimethine spacer, as in 1,3-bis-(1-pyrenyl)propane (B311, Section 13.5). Pyrene excimer
formation is commonly exploited for assaying membrane fusion 32,33 (Lipid-Mixing Assays of
Membrane Fusion—Note 13.1) and for detecting lipid domain formation. 34–36 Pyrene fatty acids are metabolically incorporated into phospholipids, di- and tri-acylglycerols and cholesteryl
esters by live cells.19,37,38
Other uses of pyrene fatty acids include:
1
2
3
4
350
•
•
•
•
•
Detecting lipid–protein interactions 9,40
Inducing photodynamic damage 41,42
Investigating phospholipase A 2 action on lipid assemblies 43–45
Studying lipid transport mechanisms and transfer proteins 46–48
Synthesizing fluorescent sphingolipid probes 49–52
Dansyl Undecanoic Acid
Dansyl undecanoic acid (DAUDA, D94; Figure 13.2.11) incorporates a polar, environmentsensitive dansyl fluorophore that preferentially locates in the polar headgroup region of lipid
bilayer membranes.53 DAUDA exhibits a 60-fold fluorescence enhancement and a large emission
spectral shift to shorter wavelengths on binding to certain proteins.54 This property has been
exploited to analyze fatty acid–binding proteins 54–57 and also to develop a fluorometric phospholipase A 2 assay (Section 17.4) based on competitive fatty acid displacement.58–61
400
450
500
550
600
Wavelength (nm)
Figure 13.2.10 Excimer formation by pyrene in
ethanol. Spectra are normalized to the 371.5 nm peak of the
monomer. All spectra are essentially identical below 400 nm
after normalization. Spectra are as follows: 1) 2 mM pyrene,
purged with argon to remove oxygen; 2) 2 mM pyrene, airequilibrated; 3) 0.5 mM pyrene (argon-purged); and 4) 2 µM
pyrene (argon-purged). The monomer-to-excimer ratio
(371.5 nm/470 nm) is dependent on both pyrene concentration and the excited-state lifetime, which is variable because
of quenching by oxygen.
cis-Parinaric Acid
The naturally occurring polyunsaturated fatty acid cis-parinaric acid (P36005, Figure
13.2.12) was initially developed as a membrane probe by Hudson and co-workers and published
in 1975.62 cis-Parinaric acid is the closest structural analog of intrinsic membrane lipids among
currently available fluorescent probes (Figure 13.2.1). The chemical and physical properties of
cis-parinaric acid have been well characterized. The lowest absorption band of cis-parinaric acid
has two main peaks around 300 nm and 320 nm, with a high extinction coefficient. cis-Parinaric
acid offers several experimentally advantageous optical properties, including a very large fluorescence Stokes shift (~100 nm) and an almost complete lack of fluorescence in water. In addition,
the fluorescence decay lifetime of cis-parinaric acid varies from 1 to ~40 nanoseconds, depending on the molecular packing density in phospholipid bilayers. Consequently, minutely detailed
information on lipid-bilayer dynamics can be obtained.
Figure 13.2.11 11-((5-Dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid (DAUDA, D94).
H
CH3CH2
C C
H
H
C C
H
H
C C
H
H
C C
O
(CH2)� C OH
H
Figure 13.2.12 cis-Parinaric acid (P36005).
™
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Probes
and
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Technologies
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551
Chapter 13 — Probes for Lipids and Membranes
Section 13.2 Fatty Acid Analogs and Phospholipids
NOTE 13.1
Lipid-Mixing Assays of Membrane Fusion
Fluorometric methods for assaying membrane fusion exploit processes,
such as nonradiative energy transfer, fluorescence quenching and pyrene
excimer formation, that are dependent on probe concentration.1–8 Assays of
membrane fusion report either the mixing of membrane lipids (described here)
or the mixing of the aqueous contents of the fused entities (Assays of Volume
Change, Membrane Fusion and Membrane Permeability—Note 14.3). Chapter
13 describes additional methods for detecting membrane fusion based on
image analysis.
NBD–Rhodamine Energy Transfer
Principle: Struck, Hoekstra and Pagano introduced lipid-mixing assays based
on NBD–rhodamine energy transfer.9 In this method (Figure 1), membranes
labeled with a combination of fluorescence energy transfer donor and acceptor lipid probes—typically NBD-PE and N-Rh-PE (N360, L1392; Section 13.2),
respectively—are mixed with unlabeled membranes. Fluorescence resonance
energy transfer (FRET), detected as rhodamine emission at ~585 nm resulting
from NBD excitation at ~470 nm, decreases when the average spatial separation
of the probes is increased upon fusion of labeled membranes with unlabeled
membranes. The reverse detection scheme, in which FRET increases upon fusion of membranes that have been separately labeled with donor and acceptor
probes, has also proven to be a useful lipid-mixing assay.10
Applications: Applications of the NBD–rhodamine assay are described in
footnoted references.11–20
Figure 1 Pictorial representation of a lipid-mixing assay based on fluorescence resonance energy transfer (FRET). The average spatial separation of
the donor (D) and acceptor (A) lipid probes increases upon fusion of labeled
membranes with unlabeled membranes, resulting in decreased efficiency
of proximity-dependent FRET (represented by yellow arrows). Decreased
FRET efficiency is registered by increased donor fluorescence intensity and
decreased acceptor fluorescence intensity.
Octadecyl Rhodamine B Self-Quenching
Principle: Lipid-mixing assays based on self-quenching of octadecyl
rhodamine B (R18, O246; Section 13.5) were originally described by Hoekstra
and co-workers.21 Octadecyl rhodamine B self-quenching occurs when the
probe is incorporated into membrane lipids at concentrations of 1–10 mole
percent.22 Unlike phospholipid analogs, octadecyl rhodamine B can readily be
introduced into existing membranes in large amounts. Fusion with unlabeled
membranes results in dilution of the probe, which is accompanied by increasing
fluorescence 23,24 (excitation/emission maxima 560/590 nm) (Figure 2). The assay
may be compromised by effects such as spontaneous transfer of the probe to
unlabeled membranes, quenching of fluorescence by proteins and probe-related
inactivation of viruses; the prevalence of these effects is currently debated.25–27
Applications: The octadecyl rhodamine B self-quenching assay is extensively used for detecting virus–cell fusion.28–39
Pyrene Excimer Formation
Principle: Pyrene-labeled fatty acids (e.g., P31, P96, P243; Section 13.2) can
be biosynthetically incorporated into viruses and cells in sufficient quantities to
produce the degree of labeling required for long-wavelength pyrene excimer
fluorescence (Figure 3). This excimer fluorescence is diminished upon fusion
of labeled membranes with unlabeled membranes (Figure 4). Fusion can be
monitored by following the increase in the ratio of monomer (~400 nm) to
excimer (~470 nm) emission, with excitation at about 340 nm. This method appears to circumvent some of the potential artifacts of the octadecyl rhodamine
B self-quenching technique 26 and, therefore, provides a useful alternative for
virus–cell fusion applications.
Applications: Applications of pyrene excimer assays for membrane fusion
are described in the footnoted references.26,28,40–43
Figure 2 Pictorial representation of a lipid-mixing assay based on fluorescence self-quenching. Fluorescence of octadecyl rhodamine B (O246),
incorporated at >1:100 with respect to host membrane lipids, is quenched
due to dye–dye interactions. Fusion with unlabeled membranes causes
dispersion of the probe, resulting in a fluorescence increase that is represented here by a color change from black to green.
O
O
O
O
O
OO
+
Pyrene excimer fluorescence
~470 nm
O O
O O
O O
O
Fusion
Pyrene monomer fluorescence
~400 nm
Figure 4 Pictorial representation of a lipid-mixing assay based on pyrene
excimer formation. Locally concentrated pyrene-labeled lipid probes
emit red-shifted fluorescence due to formation of excimers (excited-state
dimers). Probe dilution by unlabeled lipids as a result of membrane fusion
is registered by the replacement of excimer emission by blue-shifted
monomer fluorescence.
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O
O
IMPORTANT
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coveredare
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onto
IMPORTANT
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or more
Limited
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refer
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the Appendix on
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Chapter 13 — Probes for Lipids and Membranes
Section 13.2 Fatty Acid Analogs and Phospholipids
• Measurement of peroxidation in lipoproteins 63–65 and the relationship of peroxidation to
cytotoxicity 66,67 and apoptosis 68–71
• Evaluation of antioxidants 72–75
• Detection of lipoproteins following chromatographic separation 76 and structural characterization of lipoproteins 77
• Detection of lipid–protein interactions 78–80 and lipid clustering 81
• High-affinity binding to a hydrophobic pocket between the heavy chain of myosin subfragment-1 and its essential light chain 82
• Investigation of the mechanism of fatty acid–binding proteins 83–85 and phospholipid-transfer proteins 86,87
1
2
3
4
350
400
450
500
550
600
Wavelength (nm)
Figure 3 Excimer formation by pyrene in
ethanol. Spectra are normalized to the 371.5 nm
peak of the monomer. All spectra are essentially
identical below 400 nm after normalization. Spectra
are as follows: 1) 2 mM pyrene, purged with argon
to remove oxygen; 2) 2 mM pyrene, air-equilibrated;
3) 0.5 mM pyrene (argon-purged); and 4) 2 µM
pyrene (argon-purged). The monomer-to-excimer
ratio (371.5 nm/470 nm) is dependent on both
pyrene concentration and the excited-state lifetime,
which is variable because of quenching by oxygen.
1. Chem Phys Lipids (2002) 116:39; 2. Anal Biochem
(2009) 386:91; 3. Methods Enzymol (1993) 220:3;
4. Methods Enzymol (1993) 220:15; 5. Proc Natl
Acad Sci U S A (2009) 106:979; 6. Annu Rev Biophys
Biophys Chem (1989) 18:187; 7. Hepatology (1990)
12:61S-66S; 8. Biochemistry (1987) 26:8435;
9. Biochemistry (1981) 20:4093; 10. Methods
Enzymol (1993) 221:239; 11. Biochemistry (1994)
33:12615; 12. Biochemistry (1994) 33:5805;
13. Biochemistry (1994) 33:3201; 14. Biophys
J (1994) 67:1117; 15. J Biol Chem (1994) 269:15124;
16. J Biol Chem (1994) 269:4050; 17. J Biol Chem
(1993) 268:1716; 18. Biochemistry (1992) 31:2629;
19. Biochemistry (1991) 30:5319; 20. J Biol Chem
(1991) 266:3252; 21. Biochemistry (1984) 23:5675;
22. J Biol Chem (1990) 265:13533; 23. Biophys J (1993)
65:325; 24. Biophys J (1990) 58:1157; 25. Biochim
Biophys Acta (1994) 1190:360; 26. Biochemistry
(1993) 32:11330; 27. Biochemistry (1993) 32:900;
28. Biochemistry (1994) 33:9110; 29. Biochemistry
(1994) 33:1977; 30. Biochim Biophys Acta (1994)
1191:375; 31. J Biol Chem (1994) 269:5467;
32. Biochem J (1993) 294:325; 33. J Biol Chem
(1993) 268:25764; 34. J Biol Chem (1993) 268:9267;
35. Virology (1993) 195:855; 36. Biochemistry (1992)
31:10108; 37. Exp Cell Res (1991) 195:137; 38. J Virol
(1991) 65:4063; 39. Biochemistry (1990) 29:4054;
40. EMBO J (1993) 12:693; 41. J Virol (1992) 66:7309;
42. Biochemistry (1988) 27:30; 43. Biochim Biophys
Acta (1986) 860:301.
The extensive unsaturation of cis-parinaric acid makes it quite susceptible to oxidation. Consequently, we offer cis-parinaric acid in a 10 mL unit size of a 3 mM solution in deoxygenated ethanol (P36005); if stored protected from light under an inert argon atmosphere
at –20°C, this stock solution should be stable for at least six months. During experiments, we
strongly advise handling cis-parinaric acid samples under inert gas and preparing solutions using degassed buffers and solvents. cis-Parinaric acid is also somewhat photolabile, undergoing
photodimerization under intense illumination, resulting in loss of fluorescence. 88
ADIFAB Fatty Acid Indicator
Fatty acid–binding proteins are small cytosolic proteins found in a variety of mammalian tissues, and studies of their physiological function frequently involve fluorescent fatty acid
probes.89 To facilitate these studies, we offer ADIFAB reagent (A3880), a dual-wavelength fluorescent indicator of free fatty acids 90–92 (Figure 13.2.13, Figure 13.2.14). ADIFAB reagent is a
conjugate of I-FABP, a rat intestinal fatty acid–binding protein with a low molecular weight
(15,000 daltons) and a high binding affinity for free fatty acids,93 and the polarity-sensitive
acrylodan fluorophore (A433, Section 2.3). It is designed to provide quantitative monitoring of
free fatty acids without resorting to separative biochemical methods.44,94,95 With appropriate
precautions, which are described in the product information sheet accompanying this product,
ADIFAB can be used to determine free fatty acid concentrations between 1 nM and >20 µM.
Ex = 390 nm
_OA
Fluorescence emission
Fluorescence emission
Selected applications of cis-parinaric acid include:
400
+OA
450
500
550
600
650
Wavelength (nm)
Figure 13.2.13 Ribbon representation of the ADIFAB free fatty acid
indicator (A3880). In the left-hand image, the fatty acid binding site of
intestinal fatty acid–binding protein (yellow) is occupied by a covalently attached acrylodan fluorophore (blue). In the right-hand image,
a fatty acid molecule (gray) binds to the protein, displacing the fluorophore (green) and producing a shift of its fluorescence emission spectrum. Image contributed by Alan Kleinfeld, FFA Sciences LLC, San Diego.
Figure 13.2.14 The free fatty acid–dependent
spectral shift of ADIFAB (A3880). Spectra shown
represent 0.2 µM ADIFAB in pH 8.0 buffer with
(+OA) and without (–OA) addition of 4.7 µM cis9-octadecenoic (oleic) acid (OA). The ratio of
fluorescence emission intensities at 505 nm and
432 nm can be quantitatively related to free fatty
acid concentrations.
™
The
Probes
Handbook:
A Guide
to Fluorescent
Probes
and
Labeling
Technologies
TheMolecular
Molecular
Probes®
Handbook:
A Guide
to Fluorescent
Probes
and
Labeling
Technologies
IMPORTANT
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or moreUse
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to Appendix
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in this
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Limited
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on on
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553
Chapter 13 — Probes for Lipids and Membranes
Section 13.2 Fatty Acid Analogs and Phospholipids
Phospholipids with BODIPY® Dye–Labeled Acyl
Chains
BODIPY® Glycerophospholipids
Figure 13.2.15 2-(4,4-Difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (β-BODIPY® 500/510 C12-HPC,
D3793).
O
CH3(CH2)1� C OCH2
H3C
F
N
B
(CH2)11 C OCH
F
O
N
O
CH2O � OCH2CH2N(CH3)3
O
H3C
Figure 13.2.16 2-(4,4-Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (β-BODIPY® FL C12-HPC,
D3792).
O
CH3(CH2)1� C OCH2
H3C
F
N
B
(CH2)�
F
N
C OCH
O
O
CH2O � OCH2CH2N(CH3)3
O
H3C
Figure 13.2.17 2-(4,4-Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (β-BODIPY® FL C5-HPC, D3803).
O
CH3(CH2)1� C OCH2
F
N
B
(CH2)�
F
N
C OCH
O
O
CH2O � OCH2CH2N(CH3)3
O
Figure 13.2.18 2-(4,4-Difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (β-BODIPY® 530/550 C5-HPC,
D3815).
O
CH3(CH2)1� C OCH2
H3C
F
N
B
F
(CH2)�
C OCH
O
N
O
CH2O � O
O
2�NH�
H3C
Figure 13.2.19 2-(4,4-Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphate, diammonium salt (β-BODIPY® FL
C5-HPA, D3805).
We offer several glycerophospholipid analogs labeled with a single greenfluorescent BODIPY® 500/510 or a BODIPY® FL fluorophore or red-orange–
fluorescent BODIPY® 530/550 fluorophore on the sn-2 acyl chain, including:
• BODIPY® 500/510 dye–labeled glycerophosphocholine (D3793, Figure
13.2.15)
• BODIPY® FL dye–labeled glycerophosphocholine (D3792, Figure 13.2.16;
D3803, Figure 13.2.17)
• BODIPY® 530/550 dye–labeled glycerophosphocholine (D3815, Figure
13.2.18)
• BODIPY® FL dye–labeled phosphatidic acid (D3805, Figure 13.2.19)
In addition, we prepare a glycerophosphocholine analog with a single
nonhydrolyzable ether-linked BODIPY® FL fluorophore on the sn-1 position
(D3771, Figure 13.2.20), as well as several doubly labeled glycerophospholipids. These doubly labeled glycerophospholipids, which are discussed in greater
detail in Section 17.4, are designed primarily for detection of phospholipase A1
and phospholipase A 2 and include:
• Glycerophosphoethanolamine with a BODIPY® FL dye–labeled sn-1 acyl
chain and a dinitrophenyl quencher–modified headgroup (PED-A1,
A10070; Figure 13.2.21)
• Glycerophosphoethanolamine with a BODIPY® FL dye–labeled sn-2
acyl chain and a dinitrophenyl quencher–modified headgroup 96 (PED6,
D23739; Figure 13.2.22)
• Glycerophosphocholine with two BODIPY® FL dye–labeled acyl chains
(bis-BODIPY® FL C11-PC, B7701; Figure 13.2.22)
• Glycerophosphocholine with a BODIPY® 558/568 dye–labeled sn-1
alkyl chain and a BODIPY® FL dye–labeled sn-2 acyl chain (Red/Green
BODIPY® PC-A2, A10072; Figure 13.2.23)
The spectral properties of BODIPY® FL dye–labeled phospholipids are
summarized in Table 13.2. Unlike the nitrobenzoxadiazole (NBD) fluorophore,
the BODIPY® FL and BODIPY® 500/510 fluorophores are intrinsically lipophilic and readily localize in the membrane’s interior.1 The fluorophore is completely inaccessible to the membrane-impermeant anti–BODIPY® FL antibody
(A5770, Section 7.4), which also recognizes the BODIPY® 500/510 derivative. As
shown in Figure 13.2.24, the emission spectrum of the BODIPY® 500/510 fluorophore is much narrower than that of the NBD fluorophore. Because both the
extinction coefficient of the BODIPY® 500/510 fluorophore and its quantum
yield in a lipophilic environment (EC ~90,000 cm–1M–1 and QY ~0.9) are much
higher than those of the NBD fluorophore (EC ~20,000 cm–1M–1 and QY ~0.3),
much less BODIPY® 500/510 dye–labeled phospholipid is required for labeling
membranes.4
H3C
N
H3C
F
B
N
F
O
(CH2)� C OCH2
CH3(CH2)5 OCH
O
O
CH2O � OCH2CH2NH C
OH
Figure 13.2.20 2-Decanoyl-1-(O-(11-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diazas-indacene-3-propionyl)amino)undecyl)-sn-glycero-3-phosphocholine (D3771).
(CH2)5NH
Figure 13.2.21 PED-A1 (N-((6-(2,4-DNP)amino)hexanoyl)-1-(BODIPY® FL C5)-2-hexyl-snglycero-3-phosphoethanolamine; A10070).
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O2N
IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on
IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
the Appendix on
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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NO2
Chapter 13 — Probes for Lipids and Membranes
H C
3
Section 13.2 Fatty Acid Analogs and Phospholipids
Quenched substrate
(bis-BODIPY® FL C11-PC)
N
F
H C
3
H C
3
F
B
B
N
N
Quenched substrate
(PED6)
O
F
(CH )
2 10
(CH )
2 10
F
O
C
OCH
C
OCH
H C
3
O
+
CH O P OCH CH N(CH )
2
2 2
33
−
O
O
N
2
B
N
Phospholipase A 2
H C
3
F
CH (CH )
3
2 14
(CH )
F
24
N
C
OCH
O N
2
2
O
C OCH
O
O
P OCH CH NH C
2 2
−
O
CH O
2
(CH ) NH
25
NO
2
Phospholipase A 2
H C
3
H C
3
O
N
F
H C
3
B
CH (CH )
3
2 14
N
F
(CH )
2 10
C OCH
Fluorescent lysophospholipid
CH O
2
2
O
+
CH O P OCH CH N(CH )
2
2 2
33
−
O
F
B
O
O
P OCH CH NH C
2 2
−
O
(CH ) NH
25
NO
2
Nonfluorescent lysophospholipid
+
N
O N
2
2
HOCH
HOCH
H C
3
C OCH
O
+
(CH )
2 10
F
H C
3
C OH
O
N
F
N
H C
3
B
F
(CH )
24
C
OH
O
N
H C
3
Fluorescent fatty acid (BODIPY® FL C11 (D3862))
Fluorescent fatty acid (BODIPY® FL C5 (D3834))
N
�
F
N
B
F
H3C
O
CH2CH2 C NH(CH2)� OCH2
F
N
B
F
(CH2)� C OCH
O
N
O
CH2O � OCH2CH2N(CH3)3
O
Fluorescence emission
Figure 13.2.22 Mechanism of phospholipase activity–linked fluorescence enhancement responses of bis-BODIPY® FL C11-PC (B7701) and PED6
(D23739). Note that enzymatic cleavage of bis-BODIPY® FL C11-PC yields two fluorescent products, whereas cleavage of PED6 yields only one.
NBD-PC (N-3787)
H3 C
Figure 13.2.23 Red/Green BODIPY® PC-A2 (1-O-(6-BODIPY® 558/568-aminohexyl)2-BODIPY® FL C5-sn-glycero-3-phosphocholine; A10072).
Figure 13.2.24 Fluorescence spectra (excitation at 475 nm) of β-BODIPY® 500/510
C12-HPC (blue line peak at 516 nm, D3793) and
NBD C12-HPC (red line peak at 545 nm, N3787)
incorporated in DOPC (dioctadecenoylglycerophosphocholine) liposomes at molar
ratios of 1:400 mole:mole (labeled:unlabeled
PC). The integrated intensities of the spectra
are proportional to the relative fluorescence
quantum yields of the two probes.
BODIPY®-PC (D-3793)
500
550
600
650
Wavelength (nm)
Table 13.2 Spectral properties of some lipid probes.
Spectral Property
Pyrene
DPH
NBD
BODIPY® FL
Ex/Em (nm) *
340/376
360/430
470/530
507/513
QY (τ) †
0.6 (>100 nanoseconds)
0.8 (4–8 nanoseconds)
0.32 (5–10 nanoseconds)
0.9 (6 nanoseconds)
Concentration dependence
Excimer emission (~470 nm) at high
concentrations.
Self-quenched at high
concentrations.
Self-quenched at high
concentrations.
Excimer emission (~620 nm) at high
concentrations.
Environmental sensitivity
Very sensitive to quenching by
oxygen. Essentially nonfluorescent
in water.
Essentially
nonfluorescent in water.
Essentially nonfluorescent
in water.
Relatively insensitive. Strongly fluorescent in
both aqueous and lipid environments.
* Typical fluorescence excitation and emission maxima for membrane-intercalated probes. † QY = fluorescence quantum yield; τ = fluorescence decay lifetime. Typical values for membraneintercalated probes are listed. These values may show significant environment-dependent variations.
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Chapter 13 — Probes for Lipids and Membranes
B
1:10
Fluorescence emission
Fluorescence emission
A
500
600
Section 13.2 Fatty Acid Analogs and Phospholipids
1:5
700
500
Wavelength (nm)
600
700
Wavelength (nm)
Figure 13.2.25 A) Fluorescence spectrum of β-C8-BODIPY® 500/510 C5-HPC (D3795)
incorporated in DOPC (dioctadecenoylphosphocholine) liposomes at 1:100 mole:mole
(labeled:unlabeled PC). B) Fluorescence spectra at high molar incorporation levels: 1:10
mole:mole and 1:5 mole:mole.
Incorporation of high molar ratios (>10 mole %) of the BODIPY®
500/510 dye–labeled phospholipids into membranes results in a dramatic
spectral shift of the fluorescence emission spectrum to longer wavelengths
(Figure 13.2.25). We have also observed this spectral shift in the Golgi of
live cells that have been labeled with our BODIPY® dye–labeled ceramides
(Section 12.4) and with BODIPY® fatty acids that have been metabolically
incorporated by cells (Figure 13.2.2). In fluorescence resonance energy
transfer (FRET) measurements, the green-fluorescent BODIPY® 500/510
dye is an excellent donor to longer-wavelength BODIPY® probes 97,98
(Figure 13.2.26) and acceptor from dansyl-, DPH- or pyrene-labeled
phospholipids.99 These probe combinations offer several alternatives to
the widely used NBD–rhodamine fluorophore pair for researchers using
FRET techniques to study lipid transfer and membrane fusion.97
Applications
Once cells are labeled with a BODIPY® phospholipid, the probe
shows little tendency to spontaneously transfer between cells.100
Consequently, BODIPY® dye–labeled phospholipids have been used in
a number of studies of cell membrane structure and properties:
Fluorescence emission
1
2
5
3
4
3
4
2
5
500
1
550
600
650
Wavelength (nm)
Figure 13.2.26 Fluorescence resonance energy transfer from β-BODIPY® 500/510 C12-HPC
(peak at 516 nm, D3793) to BODIPY® 558/568 C12 (peak at 572 nm, D3835) in DOPC (dioctadecenoylglycerophosphocholine) lipid bilayers using 475 nm excitation. Ratio of acceptors to
donors is: 1) 0; 2) 0.2; 3) 0.4; 4) 0.8; and 5) 2.0.
Figure 13.2.27 Confocal laser-scanning microscopy images of a giant unilamellar phospholipid vesicle (GUV). The lipid composition of this GUV was DPPC/DLPC = 1/1, with DiIC20(3)
and β-BODIPY® FL C5-HPC (D3803) dyes at mole fraction ~0.001. Excitation was at 488 nm. The
upper left image is the fluorescence emission through a 585 nm longpass filter, thus almost
exclusively from DiIC20(3). The lower left image is the emission through a 522 ± 35 nm bandpass filter, thus almost exclusively from β-BODIPY® FL C5-HPC. The right image is color-merged,
using the public domain NIH Image program. Image contributed by Gerald W. Feigenson,
Cornell University, and reprinted with permission from Biophys J (2001) 80:2775.
• Despite their good photostability, BODIPY® lipids are useful for
fluorescence recovery after photobleaching (FRAP) measurements
of lipid diffusion.101,102
• Researchers have used BODIPY® fatty acids and phospholipids to visualize compartmentalization of specific lipid classes in
Schistosoma mansoni 103 and fungi.104,105
• β-BODIPY® FL C12-HPC (D3792) has been used to examine lipid–protein interactions involved in bacterial protein secretion via
fluorescence resonance energy transfer (FRET) measurements 106
(Fluorescence Resonance Energy Transfer (FRET)—Note 1.2).
• β-BODIPY® FL C5-HPC 107 (D3803) has been used to characterize lipid domains by fluorescence correlation spectroscopy 108
(Fluorescence Correlation Spectroscopy (FCS)—Note 1.3), confocal laser-scanning microscopy 109 (Figure 13.2.27) and near-field
scanning optical microscopy.101,110
• bis-BODIPY® FL C11-PC (B7701) has BODIPY® FL dye–labeled
sn-1 and sn-2 acyl groups (Figure 13.2.28), resulting in partially
quenched fluorescence that increases when one of the acyl groups is
hydrolyzed by phospholipase A1 or A 2. The hydrolysis products are
BODIPY® FL undecanoic acid (D3862) and BODIPY® FL dye–labeled lysophosphatidylcholine (Figure 13.2.22). The probe has been
used successfully in human neutrophils, plants and zebrafish to
detect phospholipase A activity 111–116 (Section 17.4).
• β-BODIPY® FL C5-HPC (D3803) has been used to investigate the
cellular uptake of antineoplastic ether lipids.117
Figure 13.2.28 1,2-bis-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoyl)sn-glycero-3-phosphocholine (bis-BODIPY® FL C11-PC, B7701).
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Chapter 13 — Probes for Lipids and Membranes
Section 13.2 Fatty Acid Analogs and Phospholipids
Phospholipid with DPH-Labeled Acyl Chain
Properties
The fluorescent phospholipid analog β-DPH HPC (D476) comprises
diphenylhexatriene propionic acid coupled to glycerophosphocholine at
the sn-2 position (Figure 13.2.29). It is therefore related to the neutral
membrane probe DPH and the cationic derivative TMA-DPH (D202,
T204; Section 13.5). DPH and its derivatives exhibit strong fluorescence
enhancement when incorporated into membranes, as well as sensitive fluorescence polarization (anisotropy) responses to lipid ordering
(Fluorescence Polarization (FP)—Note 1.4). β-DPH HPC was originally
devised to improve the localization of DPH in membranes.118,119 Unlike
underivatized DPH, it can be used to specifically label one leaflet of a
lipid bilayer, facilitating analysis of membrane asymmetry.120
Applications
DPH derivatives are predominantly used to investigate the structure and dynamics of the membrane interior either by fluorescence
polarization or lifetime measurements. Researchers have used β-DPH
HPC as a probe for lipid–protein interactions,121–123 alcohol-induced
perturbations of membrane structure,124,125 molecular organization and dynamics of lipid bilayers 11,126–128 and lipid peroxidation.129
Fluorescence lifetime measurements of β-DPH HPC provide a sensitive indicator of membrane fusion.130–132 In addition to membrane
fusion, β-DPH HPC has been used to monitor various other lipidtransfer processes.133–135
Applications
NBD acyl–modified probes are used for investigating lipid traffic, either by directly visualizing NBD fluorescence,152–155 by exploiting NBD self-quenching 156–158 or by fluorescence resonance energy
transfer methods.140,152,159–161 Lateral domains in model monolayers,
bilayers and cell membranes have been characterized using NBD phospholipids in conjunction with fluorescence recovery after photobleaching 162–164 (FRAP), fluorescence resonance energy transfer 165 (FRET)
(Fluorescence Resonance Energy Transfer (FRET)—Note 1.2) and direct microscopy techniques.166–169 Transmembrane lipid distribution
(Lipid-Mixing Assays of Membrane Fusion—Note 13.1) has been assessed using fluorescence resonance energy transfer from NBD HPC
to rhodamine DHPE 149,151,170 (L1392) or alternatively by selective dithionite (S2O42–) reduction of NBD phospholipids in the outer membrane
monolayer 171 (Figure 13.2.31).
Figure 13.2.29 β-DPH HPC (2-(3-(diphenylhexatrienyl)propanoyl)-1-hexadecanoyl-sn-glycero3-phosphocholine; D476).
Phospholipids with NBD-Labeled
Acyl Chains
Properties
Our acyl-modified nitrobenzoxadiazole (NBD) phospholipid
probes include both the NBD hexanoyl- and NBD dodecanoyl-glycerophosphocholines (NBD C6 -HPC, N3786; Figure 13.2.30 and NBD
C12-HPC, N3787). Table 13.2 compares the spectral properties of these
probes with those of the BODIPY®, DPH and pyrene lipid probes. Unlike
the BODIPY® phospholipids, the location of the relatively polar NBD
fluorophore of NBD C6 -HPC and NBD C12-HPC in phospholipid bilayers does not appear to conform to expectations based on the probe
structure. A variety of physical evidence indicates that the NBD moiety
"loops back" to the head-group region 136–139 (Figure 13.2.1). In fact, the
fluorophore in this acyl-modified phospholipid appears to probe the
same location as does the head group–labeled glycerophosphoethanolamine derivative NBD-PE 17 (N360).
These NBD probes transfer spontaneously between membranes,
with NBD C6 -HPC transferring more rapidly than its more lipophilic C12 analog.140,141 NBD C6 -HPC can be readily removed (backexchanged) from the plasma membrane by incubating the labeled
cells either with unlabeled lipid vesicles 142 or with bovine serum albumin.143–145 This property is useful for quantitating lipid transfer and
for studying phospholipid distribution asymmetry and transmembrane
"flip-flop" rates in lipid bilayers.17,146–151
Figure 13.2.30 2-(6-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-snglycero-3-phosphocholine (NBD C6-HPC, N3786).
O
NH(CH )
25
N
C
O
OH
2−
S O
2 4
NH(CH )
25
O
Fluorescent
OH
O
N
NO
2
C
N
N
NH
2
Nonfluorescent
Figure 13.2.31 Dithionite reduction of 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid (NBD-X, N316). The elimination of fluorescence associated with this reaction, coupled
with the fact that extraneously added dithionite is not membrane permeant, can be used to
determine whether the NBD fluorophore is located in the external or internal monolayer of
lipid bilayer membranes.
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IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on
IMPORTANT NOTICE
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Chapter 13 — Probes for Lipids and Membranes
Section 13.2 Fatty Acid Analogs and Phospholipids
Phospholipids with Pyrene-Labeled Acyl Chains
Properties
Figure 13.2.32 1-Hexadecanoyl-2-(1-pyrenedecanoyl)-snglycero-3-phosphocholine (β-py-C10-HPC, H361).
Phospholipid analogs with pyrene-labeled sn-2 acyl chains (Figure 13.2.32) are among
the most popular fluorescent membrane probes.29,172,173 We offer pyrenedecanoyl-labeled
glycerophospholipids with phosphocholine (H361) and phosphoglycerol (H3809) head
groups.
The spectral properties of the pyrene lipid probes are summarized in Table 13.2. Of
primary importance in terms of practical applications is the concentration-dependent formation of excited-state pyrene dimers (excimers), which exhibit a distinctive red-shifted
emission (peak ~470 nm) (Figure 13.2.10).
Applications
Figure 13.2.33 1-Hexadecanoyl-2-(1-pyrenedecanoyl)-snglycero-3-phosphoglycerol, ammonium salt (β-py-C10-PG,
H3809).
Figure 13.2.34 1,2-Bis-(1-pyrenebutanoyl)-sn-glycero-3phosphocholine (B3781).
The excimer-forming properties of pyrene are well suited for monitoring membrane
fusion (Lipid-Mixing Assays of Membrane Fusion—Note 13.1) and phospholipid transfer
processes.37,173–178 The monomer/excimer emission ratio can also be used to characterize
membrane structural domains and their dependence on temperature, lipid composition
and other external factors.179–182 Pyrenedecanoyl glycerophosphocholine (β-py-C10-HPC,
H361) has been used to elucidate the effect of extrinsic species such as Ca 2+,183 plateletactivating factor,184 drugs,185 membrane-associated proteins 186–188 and ethanol 189 on lipid
bilayer structure and dynamics. The anionic phosphoglycerol analog (H3809, Figure
13.2.33) is preferred as a substrate for secretory phosholipases A 2 relative to other phospholipid classes.190,191 The long excited-state lifetime of pyrene (Table 13.2) renders the
fluorescence of its conjugates very susceptible to oxygen quenching, and consequently
these probes can be used to measure oxygen concentrations in solutions,192 lipid bilayers 193 and cells.194,195
Glycerophospholipids in which both alcohols are esterified to pyrene fatty acids
(Figure 13.2.1), as in our bis-(1-pyrenebutanoyl)- and bis-(1-pyrenedecanoyl)glycerophosphocholines (B3781, Figure 13.2.34; B3782) show strong excimer fluorescence, with
maximum emission near 470 nm.29 Hydrolysis of either fatty acid ester by a phospholipase
results in liberation of a pyrene fatty acid and an emission shift to shorter wavelengths,
making these probes useful as phospholipase substrates 196–199 (Section 17.4).
NOTE 13.2
Antibodies for Detecting Membrane-Surface Labels
For detecting labels at the membrane surface, we offer antibodies
that recognize the following labels:
•
•
•
•
•
•
•
•
•
•
Alexa Fluor® 488 dye (A11094)
BODIPY® FL dye (A5770)
Alexa Fluor® 405 and Cascade Blue® dyes (A5760)
Dansyl (A6398)
Dinitrophenyl chromophore (A6423, A6430, A6435, A11097,
Q17421MP)
Fluorescein and Oregon Green® dyes (A889, A982, A6413, A6421,
A11090, A11091, A11095, A11096, Q15421MP, Q15431MP)
Green Fluorescent Protein (GFP, A6455, A10259, A10262, A10263,
A11120, A11121, A11122, A21311, A21312, A31851, A31852,
G10362)
Lucifer yellow (A5750, A5751)
Tetramethylrhodamine (A6397)
Texas Red® dye (A6399)
Fluorescent conjugates of several of these anti-dye and anti-hapten
antibodies are available; see Section 7.4 and Table 7.8 for complete product
information. These antibodies can be used for direct detection of labeled
phospholipids via fluorescence quenching1,2 (or fluorescence enhancement, in
the case of the anti-dansyl antibody). When used in conjunction with phospholipids with dye-labeled head groups (Table 13.1), they are important tools for:
•
•
•
Studies of molecular recognition mechanisms at membrane surfaces3
Lipid diffusion measurements4,5
Quantitation of lipid internalization by endocytosis6,7
In addition to anti-fluorophore antibodies, we offer a selection of
streptavidin conjugates (Section 7.6, Table 7.9) for detecting biotinylated
phospholipids.
1. Biochemistry (1999) 38:976; 2. J Biol Chem (1998) 273:22950; 3. Angew Chem Int Ed
Engl (1990) 29:1269; 4. J Cell Biol (1993) 120:25; 5. Proc Natl Acad Sci U S A (1991) 88:6274;
6. J Cell Biol (1988) 106:1083; 7. Cell (1991) 64:393.
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Chapter 13 — Probes for Lipids and Membranes
Section 13.2 Fatty Acid Analogs and Phospholipids
Phospholipid with an NBD-Labeled Head Group
Phospholipids with a Fluorescent or
Biotinylated Head Group
Phospholipid with a Dansyl-Labeled Head Group
The phospholipid analog incorporating the environment-sensitive 200 dansyl fluorophore (dansyl DHPE, D57; Figure 13.2.35) is a
useful probe of lipid–water interfaces.53,201 It is sensitive to the interactions of a number of proteins, including protein kinase C, 202,203 annexins 204,205 and phospholipase A 2,206–208 with membrane surfaces. Dansyl
DHPE has also been used to examine the effects of cholesterol on the
accessibility of the dansyl hapten to antibodies 209 (Antibodies for
Detecting Membrane-Surface Labels—Note 13.2).
Phospholipid with a Marina Blue®
Dye–Labeled Head Group
Marina Blue® DHPE (M12652, Figure 13.2.36) is optimally excited
by the intense 365 nm spectral line of the mercury-arc lamp and exhibits bright blue fluorescence emission near 460 nm. Significantly, the
pKa value of this 6,8-difluoro-7-hydroxycoumarin derivative is 2–3 log
units lower than that of nonfluorinated 7-hydroxycoumarin analogs;
consequently, Marina Blue® DHPE is expected to be strongly fluorescent
in membranes, even at neutral pH.
Phospholipid with a Pacific Blue™
Dye–Labeled Head Group
The Pacific Blue™ dye–labeled phospholipid (Pacific Blue™ DMPE,
P22652; Figure 13.2.37) is our only head group–labeled phospholipid
with tetradecanoyl (myristoyl) esters rather than hexadecanoyl (palmitoyl) esters. This blue-fluorescent phospholipid is structurally similar
to a phospholipid described by Gonzalez and Tsien for use in a FRETbased measurement of membrane potential.210
N(CH3)2
(CH3CH2)3NH
The widely used membrane probe nitrobenzoxadiazolyldihexadecanoylglycerophosphoethanolamine 17 (NBD-PE, N360; Figure 13.2.38) has
three important optical properties: photolability, which makes it suitable
for photobleaching recovery measurements; concentration-dependent
self-quenching; and fluorescence resonance energy transfer to rhodamine
acceptors (usually rhodamine DHPE, L1392). Spectroscopic characteristics of NBD-PE are generally similar to those described for our phospholipids with NBD-labeled acyl chains (N3786, N3787). NBD-PE is frequently used in NBD–rhodamine fluorescence energy transfer experiments to
monitor membrane fusion (Lipid-Mixing Assays of Membrane Fusion—
Note 13.1). In addition, this method can be used to detect lipid domain
formation 165 and intermembrane lipid transfer 211–214 and to determine the
transbilayer distribution of phospholipids.151 Attachment of the NBD fluorophore to the head group makes NBD-PE resistant to transfer between
vesicles.142 NBD-PE has been used in combination with either rhodamine
DHPE (L1392) or Texas Red® DHPE (T1395MP) for visualizing the spatial relationships of lipid populations by fluorescence resonance energy
transfer microscopy.215 The nitro group of NBD can be reduced with sodium dithionite, irreversibly eliminating the dye’s fluorescence (Figure
13.2.31). This technique can be employed to determine whether the probe
is localized on the outer or inner leaflet of the cell membrane.171,216–218 The
argon-ion laser–excitable NBD-PE is also a frequent choice for fluorescence recovery after photobleaching (FRAP) measurements of lateral diffusion in membranes.219–222 In addition, NBD-PE is of particular value for
monitoring bilayer-to-hexagonal phase transitions, because these transitions cause an increase in NBD-PE’s fluorescence intensity.223–225
Phospholipid with a Fluorescein-Labeled Head Group
Fluorescein-derivatized
dihexadecanoylglycerophosphoethanolamine (fluorescein DHPE, F362; Figure 13.2.39) is a membranesurface probe that is sensitive to both the local electrostatic potential
and pH.226–228 An anti–fluorescein/Oregon Green® antibody (A889,
Section 7.4) has been employed in combination with fluorescein DHPE
O
CH3(CH2)1� C OCH2
CH3(CH2)1� C OCH
O
O
�O2
CH2O � OCH2CH2NH
O
Figure 13.2.35 N-(5-dimethylaminonaphthalene-1-sulfonyl)-1,2-dihexadecanoyl-sn-glycero3-phosphoethanolamine, triethylammonium salt (dansyl DHPE, D57).
Figure 13.2.38 NBD-PE (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero3-phosphoethanolamine, triethylammonium salt; N360).
Figure 13.2.36 Marina Blue® 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine
(Marina Blue® DHPE, M12652).
(CH3CH2)3NH
O
CH3(CH2)12 C OCH2
CH3(CH2)12 C OCH
O
F
O
O
OH
O
CH2O � OCH2CH2NH C
O
F
O
Figure 13.2.37 Pacific Blue™ DMPE (Pacific Blue™ 1,2-ditetradecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt; P22652).
Figure 13.2.39 Fluorescein DHPE (N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-snglycero-3-phosphoethanolamine, triethylammonium salt; F362).
™
The
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A Guide
to Fluorescent
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and
Labeling
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A Guide
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Probes
and
Labeling
Technologies
IMPORTANT
NOTICE:described
The products
described
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UseLicense(s).
Label License(s).
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to the
Appendix
IMPORTANT NOTICE
: The products
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Chapter 13 — Probes for Lipids and Membranes
Section 13.2 Fatty Acid Analogs and Phospholipids
to investigate specific recognition interactions at membrane surfaces 229,230 (Antibodies for Detecting Membrane-Surface Labels—Note
13.2).
Because of fluorescein’s photolability, fluorescein DHPE is a useful reagent for measuring lateral diffusion in membranes using fluorescence photobleaching recovery methods.231,232 Another technique,
single-particle tracking (SPT), provides direct measurements of diffusion rates by calculating the trajectories of fluorescent polystyrene
beads or colloidal gold particles from time-sequential images.233,234
FluoSpheres® fluorescent microspheres (Section 6.5) were labeled with
streptavidin and then coupled to fluorescein DHPE using a biotinylated conjugate of anti–fluorescein/Oregon Green® monoclonal 4-4-20
(A6421, Section 7.4). Diffusion rates measured with this bridged conjugate in glass-supported phospholipid bilayers were the same as those
determined with streptavidin beads coupled directly to biotin-X DHPE
(B1616). Fluorescein DHPE has also been used in conjunction with
polyclonal anti–fluorescein/Oregon Green® antibody (A889, Section 7.4)
to prepare colloidal gold probes for SPT diffusion measurements in supported phospholipid bilayers and in keratocyte plasma membranes.235
Phospholipid with an Oregon Green®
488 Dye–Labeled Head Group
With absorption and emission spectra that are virtually superimposable on those of fluorescein, our Oregon Green® 488 DHPE (O12650,
Figure 13.2.40) provides an important alternative to fluorescein DHPE
in its many applications. When compared with the fluorescein derivative, Oregon Green® 488 DHPE exhibits greater photostability and a
lower pKa (pKa = 4.7 versus 6.4 for fluorescein); however, these pKa values may differ when the probes are bound to membranes.
Phospholipid with a BODIPY® FL
Dye–Labeled Head Group
Our phospholipid with the green-fluorescent BODIPY® FL dye attached to the head group (BODIPY® FL DHPE, D3800; Figure 13.2.41)
has significant potential for studies of molecular recognition interactions at membrane surfaces (Antibodies for Detecting MembraneSurface Labels—Note 13.2). Spectral properties of this BODIPY® probe
is generally the same as those described above for phospholipids with
BODIPY® FL dye–labeled acyl chains.
Phospholipids with a Rhodamine or Texas
Red® Dye–Labeled Head Group
The rhodamine-labeled phospholipids TRITC DHPE (T1391, Figure
13.2.42) and rhodamine DHPE (often referred to as N-Rh-PE, L1392;
Figure 13.2.1) do not readily transfer between separated lipid bilayers.140,236 This property has led to the extensive use of rhodamine DHPE
for membrane fusion assays based on fluorescence resonance energy
transfer from NBD-PE (Lipid-Mixing Assays of Membrane Fusion—
Note 13.1). In addition, these probes are good resonance energy transfer
acceptors from fluorescent lipid analogs such as the BODIPY® and NBD
phospholipids 237 and from protein labels such as 5-iodoacetamidofluorescein 5-IAF, I30451; Section 2.2) and IAEDANS 238,239 (I14, Section
2.3). Rhodamine-labeled phospholipids have also been used as tracers
for membrane traffic during endocytosis 240 and for lipid processing in
hepatocytes.241 Texas Red® DHPE (T1395MP) is principally employed
as an energy transfer acceptor from NBD, BODIPY® and fluorescein
lipid probes. The longer emission wavelength of the Texas Red® dye provides superior separation of the donor and acceptor emission signals in
resonance energy transfer microscopy.216,242 This technique has enabled
visualization of ATP-dependent fusion of liposomes with the Golgi apparatus.243 Membrane flux during hemagglutinin-mediated cell–cell
fusion has been visualized using Texas Red® DHPE and the lipophilic
carbocyanine DiI (D282, D3911; Section 13.4) as membrane labels.244
Phospholipids with a Biotinylated Head Group
We offer phospholipids labeled with a biotin at the head group to
facilitate binding of labeled membranes to other biomolecules. The biotinylated phospholipids (biotin DHPE, B1550; biotin-X DHPE, B1616,
Figure 13.2.43) can be used to couple avidin or streptavidin (Table 7.9)
to cell membranes, liposomes and lipid monolayers.245–248 Avidin can
then be employed as a bridge for antibody coupling or for assembling
liposomes into multiplex structures.249,250 Liposomes incorporating
biotinylated phospholipids can also be used to immobilize membranebound proteins for analysis by affinity chromatography.251 Interactions
of biotinylated lipids with streptavidin provide a model for molecular
(CH3)2N
O
(CH3CH2)3NH
CH3(CH2)�� C OCH2
CH3(CH2)�� C OCH
O
N(CH3)2
C O
O
CH2O � OCH2CH2NH C NH
O
O
O
�
Figure 13.2.42 TRITC DHPE (N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt; T1391).
Figure 13.2.40 Oregon Green® 488 DHPE (Oregon Green® 488 1,2-dihexadecanoyl-sn-glycero3-phosphoethanolamine; O12650).
(CH3CH2)3NH
O
CH3(CH2)�� C OCH2
CH3(CH2)�� C OCH
O
CH3
N
O
CH2O � OCH2CH2NH C CH2CH2
O
F
B
N
F
CH3
O
Figure 13.2.41 N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (BODIPY®
FL DHPE, D3800).
Figure 13.2.43 Biotin-X DHPE (N-((6-(biotinoyl)amino)hexanoyl)-1,2-dihexadecanoyl-snglycero-3-phosphoethanolamine, triethylammonium salt; B1616).
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are
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or more
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Chapter 13 — Probes for Lipids and Membranes
Section 13.2 Fatty Acid Analogs and Phospholipids
recognition processes at membrane surfaces.252–254 The phase structure
of lipid assemblies incorporating biotinylated phospholipids has been
studied by X-ray diffraction,218 31P NMR and differential scanning
calorimetry.255,256
LipidTOX™ Phospholipid and Neutral Lipid
Stains for High-Content Screening
With the resolution inherent in an image-based methodology and
the productivity of high-throughput assays, high-content screening
(HCS) or automated imaging provides a powerful tool for studying
biology in a spatial and temporal context. Using HCS technology, researchers can examine multiple cellular targets and parameters in a
large number of individually imaged cells and quantitatively assess the
data. While many Molecular Probes® products can be directly applied
to HCS protocols, we have developed validated tools and assays specifically for HCS platforms. These HCS products are:
• Validated on multiple imaging platforms
• Packaged in automation-compatible formulations
• Compatible with multiplex applications
Although designed for HCS platforms, HCS products and kits can
also be used with conventional fluorescence microscopes equipped with
standard optical filter sets.
HCS LipidTOX™ Phospholipidosis Detection Reagents
Phospholipidosis is often triggered by cationic amphiphilic drugs,
which become enriched in lysosomes to high concentrations and inhibit normal metabolism of phospholipids. The subsequent intracellular accumulation of phospholipids and formation of lamellar bodies—
phospholipidosis—can be detected in cells incubated in the presence of
phospholipids conjugated to fluorescent dyes.
HCS LipidTOX™ Green and HCS LipidTOX™ Red phospholipidosis
detection reagents (H34350, H34351), also called LipidTOX™ phospholipid stains, were specifically developed to characterize the potentially
toxic side effects of compounds on lipid metabolism in mammalian cell
lines using image-based HCS assays.257 Key advantages of this series of
phospholipidosis detection reagents over conventional stains such as
NBD-PE (N360) include their ready-to-use aqueous formulation, their
narrow emission profiles (excitation/emission maxima ~495/525 nm
for HCS LipidTOX™ Green phospholipidosis detection reagent and
~595/615 nm for HCS LipidTOX™ Red phospholipidosis detection reagent) and their compatibility with HCS LipidTOX™ neutral lipid stains.
HCS LipidTOX™ phospholipidosis detection reagents have not been
observed to affect the normal growth of cells, and their live-cell staining
patterns are maintained after formaldehyde fixation. These reagents are
designed for fixed–end point workflows in which formaldehyde-fixed
cells in microplates are processed, imaged and analyzed. HCS LipidTOX™
phospholipidosis detection reagents can easily be detected with fluorescence microscopes or HCS readers equipped with standard filter sets.
HCS LipidTOX™ Neutral Lipid Stains
As with phospholipidosis, steatosis or the intracellular accumulation of neutral lipids as lipid droplets or globules is often triggered by
drugs that affect the metabolism of fatty acids or neutral lipids. HCS
LipidTOX™ neutral lipid stains were developed to characterize the effects of drugs and other compounds on lipid metabolism in mammalian cell lines. HCS LipidTOX™ neutral lipid stains have an extremely
high affinity for neutral lipid droplets. These reagents are added after
cell fixation and do not require subsequent wash steps after incubation with the sample. Key advantages of this series of neutral lipid
stains over conventional stains such as nile red (N1142; Section 13.5)
include their ready-to-use formulations, their flexibility for multiplexing protocols and their compatibility with HCS LipidTOX™
phospholipidosis detection reagents. HCS LipidTOX™ neutral lipid
stains can also be used to monitor the formation and differentiation of adipocytes, a process called adipogenesis. Adipogenesis is of
acute interest to the biomedical and drug discovery community as
it plays an important role in diseases such as obesity, diabetes and
atherosclerosis.
Described more thoroughly in Section 13.5, HCS LipidTOX™ neutral lipid stains are available with green, red and deep red fluorescence
emission:
• HCS LipidTOX™ Green neutral lipid stain (H34475), with excitation/emission maxima ~495/505 nm (Figure 13.2.44)
• HCS LipidTOX™ Red neutral lipid stain (H34476), with excitation/
emission maxima ~577/609 nm
• HCS LipidTOX™ Deep Red neutral lipid stain (H34477), with excitation/emission maxima ~637/655 nm
HCS LipidTOX™ neutral lipid stains are designed for fixed–end
point workflows in which formaldehyde-fixed cells in microplates are
processed, imaged and analyzed. These stains can easily be detected
with fluorescence microscopes or HCS readers equipped with standard
filter sets.
Figure 13.2.44 LipidTOX™ Green neutral lipid stain and fatty acid–binding protein (FABP4)
antibody labeling in adipocytes. Adipocytes differentiated from 3T3-L1 mouse fibroblasts were fixed with formaldehyde and permeabilized with saponin before labeling with
rabbit anti–fatty acid binding protein (FABP4) IgG (red). These cells were then stained with
LipidTOX™ Green neutral lipid stain (H34475, green), counterstained with DAPI (D1306,
D21490; blue) and mounted in ProLong® Gold antifade reagent (P36930).
™
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Handbook:
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TheMolecular
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Handbook:
A Guide
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Probes
and
Labeling
Technologies
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Label License(s).
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manual
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Label
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onon
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561
Chapter 13 — Probes for Lipids and Membranes
Section 13.2 Fatty Acid Analogs and Phospholipids
HCS LipidTOX™ Phospholipidosis
and Steatosis Detection Kit
The detection and analysis of prelethal mechanisms in toxicological
profiling and compound screening are extremely important components of the drug discovery process. The cationic amphiphilic drugs
are among the most prominent examples of compounds that impact
lipid metabolism of cells. These drugs tend to become enriched in lysosomes to high concentrations and inhibit the normal metabolism of
phospholipids, which in turn causes the intracellular accumulation of
phospholipids and the formation of lamellar bodies. Other drug classes
more adversely affect various aspects of fatty acid or neutral lipid metabolism, leading to the cytoplasmic accumulation of neutral lipid as
lipid droplets or globules.
The HCS LipidTOX™ Phospholipidosis and Steatosis Detection Kit
(H34157, H34158) provides a complete set of reagents for performing
validated HCS assays to detect and distinguish these two facets of cytotoxicity—the intracellular accumulation of phospholipids (phospholipidosis) and of neutral lipids (steatosis)—in mammalian cell lines after
exposure to test compounds.258 This kit includes an aqueous, red-fluorescent formulation of labeled phospholipids (LipidTOX™ Red phospholipid stain, excitation/emission ~595/615 nm) and a ready-to-use,
highly selective green-fluorescent stain for neutral lipids (LipidTOX™
Green neutral lipid stain, excitation/emission ~495/505 nm), which can
be used sequentially for the analysis of phospholipidosis and steatosis,
respectively, or can be used separately for single-parameter analysis.
After incubation with LipidTOX™ Red phospholipid stain and
a test compound, the cells are fixed with formaldehyde and labeled
with LipidTOX™ Green neutral lipid stain (Figure 13.2.45). Neither
LipidTOX™ Red phospholipid stain, nor LipidTOX™ Green phospholipid stain described above, requires sonication or organic solvents. Furthermore, LipidTOX™ Green neutral lipid stain (as well as
the other LipidTOX™ neutral lipid stains described above) is more selective than nile red, allowing you to easily distinguish neutral lipids
(such as those in adipocytes and cells undergoing steatosis) from other
types of lipids.
Figure 13.2.45 Multiplex detection of phospholipidosis and steatosis in HepG2 cells using
the HCS LipidTOX™ Phospholipidosis and Steatosis Detection Kit (H34157, H34158). HepG2
cells were co-incubated with tamoxifen and LipidTOX™ Red phospholipid stain, followed by
fixation with formaldehyde and labeling with HCS LipidTOX™ Green neutral lipid stain and
Hoechst 33342 (H1399, H3570, H21492).
Each HCS LipidTOX™ Phospholipidosis and Steatosis Detection Kit
provides:
•
•
•
•
LipidTOX™ Red phospholipid stain
LipidTOX™ Green neutral lipid stain
Hoechst 33342 for nuclear labeling
Propranolol, a positive-control compound for inducing
phospholipidosis
• Cyclosporin A, a positive-control compound for inducing steatosis
• Dimethylsulfoxide (DMSO)
• Detailed protocols
Sufficient reagents are provided for 240 assays (H34157, 2-plate size)
or 1200 assays (H34158, 10-plate size), based on assay volumes of 100 µL
per well. These kits are designed for fixed–end point workflows in which
formaldehyde-fixed cells in microplates are processed, imaged and analyzed. The fluorescent stains used for the analysis of phospholipidosis
and steatosis can easily be detected with fluorescence microscopes or
HCS readers equipped with standard filter sets.
REFERENCES
1. Biochim Biophys Acta (1998) 1375:13; 2. Biochemistry (1992) 31:5312; 3. Chem Phys
Lipids (2002) 116:3; 4. Anal Biochem (1991) 198:228; 5. Biochem Biophys Res Commun
(1992) 187:1594; 6. J Biol Chem (2001) 276:1391; 7. J Biol Chem (1997) 272:8531; 8. Exp
Parasitol (1997) 86:133; 9. Am J Physiol (1995) 269:G842; 10. Methods Enzymol (2000)
319:603; 11. Biochim Biophys Acta (2000) 1487:61; 12. Anal Biochem (1998) 265:290;
13. FEBS Lett (1999) 453:278; 14. Free Radic Biol Med (2002) 33:473; 15. J Biochem
Biophys Methods (1997) 35:23; 16. Photochem Photobiol (1991) 54:361; 17. Chem Phys
Lipids (1990) 53:1; 18. Biochemistry (1995) 34:11919; 19. Chem Phys Lipids (1991) 58:111;
20. Biophys J (2001) 80:832; 21. J Am Chem Soc (1975) 97:4114; 22. Anal Biochem (1988)
174:101; 23. Anal Biochem (1989) 183:231; 24. Arch Biochem Biophys (1997) 341:34;
25. Exp Cell Res (1974) 89:105; 26. Adv Exp Med Biol (1975) 75:47; 27. Biochim Biophys
Acta (1980) 591:187; 28. Photochem Photobiol (1977) 26:221; 29. Chem Phys Lipids
(2002) 116:57; 30. Chem Phys Lipids (1980) 27:199; 31. Biophys J (2001) 80:832; 32. J Biol
Chem (1997) 272:3369; 33. Biochemistry (1993) 32:11330; 34. J Biol Chem (1996)
271:3085; 35. Biophys J (1991) 60:110; 36. Biochim Biophys Acta (1992) 1106:178;
37. Chem Phys Lipids (1989) 50:191; 38. J Biol Cm (1992) 267:6563; 39. Biochemistry
(1995) 34:7271; 40. Biochemistry (1995) 34:9913; 41. Biochim Biophys Acta (1998)
1402:61; 42. J Biol Chem (2000) 275:21715; 43. J Neurosci Methods (2000) 100:127;
44. Biochemistry (1998) 37:10709; 45. Biochemistry (1998) 37:14128; 46. Biochemistry
(1994) 33:15382; 47. J Biol Chem (1998) 273:27800; 48. Biophys J (1997) 72:1732;
49. Chem Phys Lipids (1993) 65:43; 50. Biochemistry (1990) 29:697; 51. Biochemistry
(1987) 26:5943; 52. Biochim Biophys Acta (1987) 918:250; 53. Biochemistry (1998)
37:4603; 54. Biochem J (1986) 238:419; 55. Biochem J (2001) 356:387; 56. Biochemistry
(1999) 38:16932; 57. Analyst (1992) 117:1859; 58. Biochim Biophys Acta (2000) 1484:195;
59. Anal Biochem (1993) 212:65; 60. Biochem J (1991) 278:843; 61. Biochem J (1990)
266:435; 62. Proc Natl Acad Sci U S A (1975) 72:1649; 63. Biochem Pharmacol (2003)
66:947; 64. Proc Natl Acad Sci U S A (1994) 91:1183; 65. Arch Biochem Biophys (1992)
297:147; 66. Biochim Biophys Acta (1997) 1330:127; 67. Neurochem Res (1997) 22:1187;
68. Biochemistry (2000) 39:127; 69. Biochemistry (1998) 37:13781; 70. Nature (1995)
378:776; 71. Cell (1993) 75:241; 72. Biochem Pharmacol (1997) 54:937; 73. Biochemistry
(1993) 32:10692; 74. J Biol Chem (1993) 268:10906; 75. Anal Biochem (1991) 196:443;
76. Clin Chem (1998) 44:2148; 77. Chem Phys Lipids (1991) 60:1; 78. Biochemistry
(1993) 32:12420; 79. Biochemistry (1991) 30:6195; 80. Biochemistry (1990) 29:6714;
81. Biochemistry (1992) 31:1816; 82. Biophys J (1997) 72:2268; 83. Biochem J (2000)
349:377; 84. Biochemistry (1994) 33:3327; 85. J Biol Chem (1993) 268:7885; 86. Lipids
(1997) 32:1201; 87. Biochemistry (1990) 29:8548; 88. Proc Natl Acad Sci USA (1980)
77:26; 89. Mol Cell Biochem (1993) 123:45; 90. Mol Cell Biochem (1999) 192:87;
91. J Biol Chem (1994) 269:23918; 92. J Biol Chem (1992) 267:23495; 93. J Biol Chem
The
MolecularProbes®
Probes Handbook:
Handbook: AAGuide
Probes and
andLabeling
LabelingTechnologies
Technologies
The
Molecular
Guide to
toFluorescent
Fluorescent Probes
™
562
IMPORTANT
NOTICE:
The products
described
in this manual
coveredare
by covered
one or more
Limited
Use Label
License(s).
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onto
IMPORTANT
NOTICE
: The products
described
in thisaremanual
by one
or more
Limited
Use Label
License(s).
refer
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
the Appendix on
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
www.invitrogen.com/probes
thermofisher.com/probes
Chapter 13 — Probes for Lipids and Membranes
Section 13.2 Fatty Acid Analogs and Phospholipids
REFERENCES—continued
(1992) 267:23534; 94. Biochemistry (1998) 37:8011; 95. J Lipid Res (1995) 36:229;
96. Anal Biochem (1999) 276:27; 97. Biochemistry (2001) 40:8292; 98. J Fluorescence
(1994) 4:295; 99. Biochem Biophys Res Commun (1995) 207:508; 100. Biochemistry
(1997) 36:8840; 101. Biophys J (1998) 74:2184; 102. Biophys J (1996) 71:2656; 103. J Cell
Sci (1993) 106:485; 104. J Biochem (Tokyo) (2001) 129:19; 105. Biochim Biophys Acta
(1999) 1438:185; 106. Biochemistry (1996) 35:3063; 107. Biophys J (2002) 83:1511;
108. Proc Natl Acad Sci U S A (1999) 96:8461; 109. Biophys J (2001) 80:2775; 110. J Cell
Biol (1995) 130:781; 111. J Biol Chem (1999) 274:19338; 112. J Biol Chem (1992)
267:21465; 113. Plant Physiol (1996) 110:979; 114. Br J Pharmacol (1998) 124:1675;
115. J Biol Chem (1997) 272:2542; 116. Biochim Biophys Acta (1999) 1448:390;
117. Biochim Biophys Acta (1998) 1390:73; 118. Biochim Biophys Acta (1982) 692:196;
119. Biochemistry (1998) 37:8180; 120. J Biol Chem (1996) 271:11627; 121. Biochemistry
(2000) 39:10928; 122. Biochemistry (1999) 38:4604; 123. Biochemistry (1997) 36:4675;
124. Biochemistry (1997) 36:10630; 125. Biochim Biophys Acta (1988) 946:85;
126. Biophys J (1997) 72:2660; 127. Biophys J (1996) 71:892; 128. Biophys J (1996)
71:878; 129. Methods Enzymol (1994) 233:459; 130. Biochemistry (1997) 36:5827;
131. Biochemistry (1997) 36:6251; 132. J Fluorescence (1994) 4:153; 133. J Biol Chem
(1996) 271:31878; 134. Biochemistry (1998) 37:16653; 135. Biochemistry (1991) 30:4193;
136. Biophys J (2001) 80:822; 137. Biochemistry (1993) 32:10826; 138. Biochim Biophys
Acta (1988) 938:24; 139. Biochemistry (1987) 26:39; 140. Biochemistry (1982) 21:1720;
141. Biochemistry (1981) 20:2783; 142. J Biol Chem (1980) 255:5404; 143. J Biol Chem
(1994) 269:22517; 144. Biochemistry (1993) 32:3714; 145. Proc Natl Acad Sci U S A
(1989) 86:9896; 146. J Biol Chem (2000) 275:23065; 147. Biophys J (2000) 78:2628;
148. Biochemistry (1998) 37:14833; 149. Biochemistry (1994) 33:6721; 150. Biochim
Biophys Acta (2007) 1768:502; 151. Biochemistry (1992) 31:2865; 152. J Cell Biol (1993)
123:1403; 153. J Cell Biol (1991) 113:235; 154. J Cell Biol (1991) 112:267; 155. J Biol Chem
(1990) 265:5337; 156. Biochim Biophys Acta (1991) 1082:255; 157. Biochemistry (1988)
27:1889; 158. J Biol Chem (1987) 262:14172; 159. Biochemistry (2001) 40:6475; 160. Am
J Physiol (1994) 267:G80; 161. J Biol Chem (1983) 258:5368; 162. Biochemistry (1994)
33:8225; 163. J Cell Biol (1991) 112:1143; 164. J Cell Biol (1987) 105:755; 165. Biophys
J (2001) 80:1819; 166. Biochemistry (1994) 33:4483; 167. Biochemistry (1993) 32:12591;
168. Chem Phys Lipids (1991) 57:227; 169. Proc Natl Acad Sci U S A (1991) 88:1364;
170. Chem Phys Lipids (1991) 57:29; 171. Biochemistry (1998) 37:15114; 172. J Fluoresc
(2007) 17:97; 173. Biophys J (2007) 92:126; 174. Biochim Biophys Acta (2000) 1487:82;
175. Biochim Biophys Acta (2000) 1467:281; 176. Biochemistry (1993) 32:11074;
177. Biochemistry (1992) 31:5912; 178. Biochemistry (1990) 29:1593; 179. Biochemistry
(2001) 40:4181; 180. Biochemistry (1998) 37:17562; 181. Biophys J (1994) 66:1981;
182. Biophys J (1992) 63:903; 183. Biochemistry (1988) 27:3433; 184. Chem Phys Lipids
(1990) 53:129; 185. J Biol Chem (1993) 268:1074; 186. Biochemistry (1993) 32:11711;
187. Biochemistry (1993) 32:5411; 188. Biochemistry (1993) 32:5373; 189. Biophys
J (1994) 66:729; 190. Biochemistry (1997) 36:14325; 191. Biochemistry (1999) 38:7803;
192. Anal Chem (1987) 59:279; 193. Biophys J (1985) 47:613; 194. J Cell Physiol (1981)
107:329; 195. Biochim Biophys Acta (1972) 279:393; 196. Anal Biochem (1981) 116:553;
197. Biochim Biophys Acta (1994) 1192:132; 198. Anal Biochem (1994) 219:1; 199. Anal
Biochem (1995) 232:7; 200. Biochim Biophys Acta (1985) 815:351; 201. Biochim Biophys
Acta (1996) 1284:191; 202. J Biol Chem (1997) 272:6167; 203. Biochemistry (1993) 32:66;
204. Biochemistry (1997) 36:8189; 205. Biochemistry (1994) 33:13231;
206. Biochemistry (2000) 39:9623; 207. Biochemistry (1998) 37:6697; 208. Biochemistry
(1998) 37:8516; 209. Biochim Biophys Acta (1992) 1104:9; 210. Chem Biol (1997) 4:269;
211. J Biol Chem (1994) 269:10517; 212. Biochemistry (1990) 29:879; 213. Biochim
Biophys Acta (1989) 981:178; 214. Biochemistry (1988) 27:3925; 215. Methods Enzymol
(1989) 171:850; 216. Biochemistry (1994) 33:9968; 217. Chem Phys Lipids (1994) 70:205;
218. Biochemistry (1993) 32:14194; 219. Biochemistry (1977) 16:3836; 220. J Cell Biol
(1993) 122:1253; 221. J Cell Biol (1991) 115:1585; 222. J Cell Biol (1991) 115:245;
223. Biophys J (1992) 63:309; 224. Biochemistry (1990) 29:2976; 225. Biochemistry
(1988) 27:3947; 226. Z Naturforsch C (2000) 55:418; 227. Biochim Biophys Acta (1998)
1374:63; 228. J Biol Chem (1999) 274:29951; 229. Biophys J (1992) 63:823; 230. J Am
Chem Soc (1981) 103:6797; 231. Biophys J (1994) 66:25; 232. J Cell Biol (1986) 103:807;
233. J Membr Biol (1993) 135:83; 234. Proc Natl Acad Sci U S A (1991) 88:6274;
235. J Cell Biol (1993) 120:25; 236. Biochemistry (1985) 24:6390; 237. Biochemistry
(1981) 20:4093; 238. J Biol Chem (1991) 266:12082; 239. Biochemistry (1990) 29:1607;
240. Eur J Cell Biol (1990) 53:173; 241. Biochem J (1992) 284:259; 242. J Cell Biol (1986)
103:1221; 243. Cell (1988) 55:797; 244. J Cell Biol (1993) 121:543; 245. J Immunol
Methods (1993) 158:183; 246. Anal Biochem (1992) 207:341; 247. Biophys J (1991)
59:387; 248. Biochim Biophys Acta (1990) 1028:73; 249. Anal Chem (2001) 73:91;
250. Science (1994) 264:1753; 251. Anal Biochem (2000) 280:94; 252. Angew Chem Int
Ed Engl (1990) 29:1269; 253. Anal Biochem (1994) 217:128; 254. Biophys J (1993)
65:2160; 255. Biophys J (1994) 66:31; 256. Biochemistry (1993) 32:9960; 257. Toxicol Sci
(2007) 99:162; 258. Cytometry A (2009) 77:231.
DATA TABLE 13.2 FATTY ACID ANALOGS AND PHOSPHOLIPIDS
Cat. No.
A3880
A10070
A10072
B1550
B1616
B3781
B3782
B3824
B7701
D57
D94
D476
D3771
D3792
D3793
D3800
D3803
D3805
D3815
D3821
D3822
D3823
D3825
D3832
D3834
MW
~15,350
880.68
986.67
1019.45
1132.61
797.88
966.20
404.31
1029.80
1026.44
434.59
782.01
854.86
895.95
881.93
1067.23
797.77
746.68
921.91
474.44
418.33
404.31
404.31
542.47
320.15
Storage
FF,L,AA
FF,D,L
FF,D,L
FF,D
FF,D
FF,D,L
FF,D,L
F,L
FF,D,L
FF,D,L
F,L
FF,D,L
FF,D,L
FF,D,L
FF,D,L
FF,D,L
FF,D,L
FF,D,L
FF,D,L
F,L
F,L
F,L
F,L
F,L
F,L
Soluble
H2O
DMSO
DMSO
see Notes
see Notes
see Notes
see Notes
DMSO
see Notes
see Notes
DMSO, EtOH
see Notes
see Notes
see Notes
see Notes
see Notes
see Notes
see Notes
see Notes
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO, MeCN
Abs
365
505
505
<300
<300
342
340
509
505
336
335
354
506
506
509
505
503
504
534
505
505
508
509
534
505
EC
10,500
92,000
85,000
75,000
62,000
101,000
123,000
4500
4800
81,000
71,000
86,000
86,000
87,000
80,000
79,000
64,000
90,000
87,000
97,000
100,000
76,000
96,000
Em
432
512
567
none
none
471
473
515
512
517
519
428
512
513
513
511
512
511
552
512
511
514
515
552
511
Solvent
H2O
MeOH
MeOH
EtOH
EtOH
MeOH
MeOH
MeOH
MeOH
MeOH
EtOH
EtOH
EtOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
Notes
1
16
17, 18
2
2
3
4
5
2, 6
2
2, 7
2
2, 5
2, 5
2, 5
2, 5
2, 5
2, 5
5
5
5
5
5
5
continued on next page
™
The
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Handbook:
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Probes
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TheMolecular
Molecular
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Handbook:
A Guide
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Probes
and
Labeling
Technologies
IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on
IMPORTANT NOTICE
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page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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563
Chapter 13 — Probes for Lipids and Membranes
Section 13.2 Fatty Acid Analogs and Phospholipids
DATA TABLE 13.2 FATTY ACID ANALOGS AND PHOSPHOLIPIDS—continued
Cat. No.
MW
Storage
Soluble
Abs
EC
Em
Solvent
Notes
D3835
472.40
F,L
DMSO
559
91,000
568
MeOH
5
D3861
504.43
F,L
DMSO
582
140,000
591
MeOH
8
D3862
404.31
F,L
DMSO
505
92,000
510
MeOH
5
D23739
1136.13
FF,D,L
DMSO
505
92,000
511
MeOH
2, 9
F362
1182.54
FF,D,L
see Notes
496
88,000
519
MeOH
2, 10
H361
850.13
FF,D,L
see Notes
342
37,000
376
MeOH
2, 11, 12
H3809
856.09
FF,D,L
see Notes
341
38,000
376
MeOH
2, 11, 12
495
84,000
525
MeOH
15
H34350
~1100
F,L
H2O
595
112,000
615
MeOH
15
H34351
~1400
F,L
H2O
L1392
1333.81
FF,D,L
see Notes
560
75,000
581
MeOH
2
M12652
944.14
FF,D,L
see Notes
365
18,000
460
MeOH
2, 10
N316
294.27
L
DMSO
467
23,000
539
MeOH
13
N360
956.25
FF,D,L
see Notes
463
21,000
536
MeOH
2, 13
N678
378.43
L
DMSO
467
24,000
536
MeOH
13
N3786
771.89
FF,D,L
see Notes
465
21,000
533
EtOH
2, 13
N3787
856.05
FF,D,L
see Notes
465
22,000
534
EtOH
2, 13
O12650
1086.25
FF,D,L
see Notes
501
85,000
526
MeOH
2, 10
P31
372.51
L
DMF, DMSO
341
43,000
377
MeOH
11, 12
P96
400.56
L
DMF, DMSO
341
44,000
377
MeOH
11, 12
P243
456.67
L
DMF, DMSO
341
43,000
377
MeOH
11, 12
P1903MP
288.35
L
DMF, DMSO
341
43,000
376
MeOH
11, 12
P22652
961.17
FF,D,L
see Notes
411
40,000
454
MeOH
2
P36005
276.42
FF,LL,AA
EtOH
304
77,000
416
MeOH
14, 15
T1391
1236.68
FF,D,L
see Notes
540
93,000
566
MeOH
2
T1395MP
1381.85
FF,D,L
see Notes
583
115,000
601
MeOH
2
For definitions of the contents of this data table, see “Using The Molecular Probes® Handbook” in the introductory pages.
Notes
1. ADIFAB fatty acid indicator is a protein conjugate with a molecular weight of approximately 15,350. Em shifts from about 432 nm to 505 nm upon binding of fatty acids. (Mol Cell Biochem (1999)
192:87)
2. Chloroform is the most generally useful solvent for preparing stock solutions of phospholipids (including sphingomyelins). Glycerophosphocholines are usually freely soluble in ethanol. Most
other glycerophospholipids (phosphoethanolamines, phosphatidic acids and phosphoglycerols) are less soluble in ethanol, but solutions up to 1–2 mg/mL should be obtainable, using sonication to aid dispersion if necessary. Labeling of cells with fluorescent phospholipids can be enhanced by addition of cyclodextrins during incubation. (J Biol Chem (1999) 274:35359)
3. Phospholipase A cleavage generates a fluorescent fatty acid (P1903MP) and a fluorescent lysophospholipid.
4. Phospholipase A cleavage generates a fluorescent fatty acid (P31) and a fluorescent lysophospholipid.
5. The absorption and fluorescence spectra of BODIPY® derivatives are relatively insensitive to the solvent.
6. Phospholipase A cleavage results in increased fluorescence with essentially no wavelength shift. The cleavage products are D3862 and a fluorescent lysophospholipid.
7. Diphenylhexatriene (DPH) and its derivatives are essentially nonfluorescent in water. Absorption and emission spectra have multiple peaks. The wavelength, resolution and relative intensity of
these peaks are environment dependent. Abs and Em values are for the most intense peak in the solvent specified.
8. Oxidation of the polyunsaturated butadienyl portion of the BODIPY® 581/591 dye results in a shift of the fluorescence emission peak from ~590 nm to ~510 nm. (Methods Enzymol (2000)
319:603, FEBS Lett (1999) 453:278)
9. Phospholipase A2 cleavage results in increased fluorescence with essentially no wavelength shift. The cleavage products are D3834 and a dinitrophenylated lysophospholipid.
10. Spectra of this compound are in methanol containing a trace of KOH.
11. Alkylpyrene fluorescence lifetimes are up to 110 nanoseconds and are very sensitive to oxygen.
12. Pyrene derivatives exhibit structured spectra. The absorption maximum is usually about 340 nm with a subsidiary peak at about 325 nm. There are also strong absorption peaks below
300 nm. The emission maximum is usually about 376 nm with a subsidiary peak at 396 nm. Excimer emission at about 470 nm may be observed at high concentrations.
13. Fluorescence of NBD and its derivatives in water is relatively weak. QY and τ increase and Em decreases in aprotic solvents and other nonpolar environments relative to water. (Biochemistry
(1977) 16:5150, Photochem Photobiol (1991) 54:361)
14. Cis-parinaric acid is highly oxygen sensitive. Use under N2 or Ar. Cis-parinaric acid is essentially nonfluorescent in water.
15. This product is supplied as a ready-made solution in the solvent indicated under "Soluble."
16. Phospholipase A1 cleavage results in increased fluorescence with essentially no wavelength shift. The cleavage products are D3834 and a dinitrophenylated lysophospholipid.
17. A10072 exhibits dual emission (Em = 510 nm and 567 nm in MeOH, 513 nm and 575 nm when incorporated in phospholipid bilayer membranes). Phospholipase A2 cleavage results in increased 510–513 nm emission and reciprocally diminshed 567–575 nm emission.
18. A10072 is also soluble at 2 mM in 2-methoxyethanol.
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Chapter 13 — Probes for Lipids and Membranes
Section 13.2 Fatty Acid Analogs and Phospholipids
PRODUCT LIST 13.2 FATTY ACID ANALOGS AND PHOSPHOLIPIDS
Quantity
Cat. No.
Product
A3880
ADIFAB fatty acid indicator
200 µg
B1550
biotin DHPE (N-(biotinoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt)
10 mg
B1616
biotin-X DHPE (N-((6-(biotinoyl)amino)hexanoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt)
B7701
1,2-bis-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoyl)-sn-glycero-3-phosphocholine (bis-BODIPY® FL C11-PC)
5 mg
100 µg
B3781
1,2-bis-(1-pyrenebutanoyl)-sn-glycero-3-phosphocholine
1 mg
B3782
1,2-bis-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine
1 mg
B3824
BODIPY® 500/510 C4, C9 (5-butyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3-nonanoic acid)
1 mg
D3771
2-decanoyl-1-(O-(11-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)undecyl)-sn-glycero-3-phosphocholine
1 mg
D3822
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (BODIPY® FL C12)
D3792
2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (β-BODIPY® FL C12-HPC)
D3821
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid (BODIPY® FL C16)
1 mg
D3834
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid (BODIPY® FL C5)
1 mg
1 mg
100 µg
D3805
2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphate, diammonium salt (β-BODIPY® FL C5-HPA)
100 µg
D3803
D3800
2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (β-BODIPY® FL C5-HPC)
N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt
(BODIPY® FL DHPE)
100 µg
D3862
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (BODIPY® FL C11)
1 mg
D3832
4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (BODIPY® 530/550 C12)
1 mg
100 µg
D3815
2-(4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (β-BODIPY® 530/550 C5-HPC)
D3823
4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (BODIPY® 500/510 C1, C12)
D3793
2-(4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (β-BODIPY® 500/510 C12-HPC)
D3825
4,4-difluoro-5-octyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid (BODIPY® 500/510 C8, C5)
1 mg
D3861
4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (BODIPY® 581/591 C11)
1 mg
D3835
4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (BODIPY® 558/568 C12)
D94
11-((5-dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid (DAUDA)
D57
D23739
N-(5-dimethylaminonaphthalene-1-sulfonyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (dansyl DHPE)
N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3phosphoethanolamine, triethylammonium salt (PED6)
D476
β-DPH HPC (2-(3-(diphenylhexatrienyl)propanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine)
1 mg
100 µg
1 mg
100 µg
1 mg
100 mg
25 mg
1 mg
F362
fluorescein DHPE (N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt)
5 mg
H34350
HCS LipidTOX™ Green phospholipidosis detection reagent *1000X aqueous solution* *for cellular imaging* *10-plate size*
each
H34157
HCS LipidTOX™ Phospholipidosis and Steatosis Detection Kit *for high-content screening* *for cellular imaging* *2-plate size*
1 kit
H34158
HCS LipidTOX™ Phospholipidosis and Steatosis Detection Kit *for high-content screening* *for cellular imaging* *10-plate size*
1 kit
H34351
HCS LipidTOX™ Red phospholipidosis detection reagent *1000X aqueous solution* *for cellular imaging* *10-plate size*
each
H361
1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (β-py-C10-HPC)
1 mg
H3809
1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphoglycerol, ammonium salt (β-py-C10-PG)
1 mg
L1392
Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (rhodamine DHPE)
5 mg
M12652
Marina Blue® 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Marina Blue® DHPE)
1 mg
N360
NBD-PE (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt)
N316
NBD-X (6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid)
100 mg
10 mg
100 mg
N678
12-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoic acid
N3787
2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD C12-HPC)
5 mg
N3786
2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD C6-HPC)
5 mg
O12650
Oregon Green® 488 DHPE (Oregon Green® 488 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine)
1 mg
P22652
Pacific Blue™ DMPE (Pacific Blue™ 1,2-ditetradecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt)
1 mg
P36005
cis-parinaric acid *3 mM in ethanol*
A10070
PED-A1 (N-((6-(2,4-DNP)amino)hexanoyl)-1-(BODIPY® FL C5)-2-hexyl-sn-glycero-3-phosphoethanolamine) *phospholipase A1 selective substrate*
100 µg
100 mg
10 mL
P1903MP
1-pyrenebutanoic acid *high purity*
P31
1-pyrenedecanoic acid
25 mg
P96
1-pyrenedodecanoic acid
25 mg
P243
1-pyrenehexadecanoic acid
A10072
Red/Green BODIPY® PC-A2 (1-O-(6-BODIPY® 558/568-aminohexyl)-2-BODIPY® FL C5-sn-glycero-3-phosphocholine) *ratiometric phospholipase A2 substrate*
5 mg
100 µg
T1395MP
Texas Red® DHPE (Texas Red® 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt)
1 mg
T1391
TRITC DHPE (N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt)
1 mg
™
The
Handbook:
A Guide
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Probes
and
TheMolecular
MolecularProbes
Probes®
Handbook:
A Guide
to Fluorescent
Probes
andLabeling
LabelingTechnologies
Technologies
IMPORTANT
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Label License(s).
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IMPORTANT NOTICE
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coveredarebycovered
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Please
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onon
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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Chapter 13 — Probes for Lipids and Membranes
Section 13.3 Sphingolipids, Steroids, Lipopolysaccharides and Related Probes
13.3 Sphingolipids, Steroids, Lipopolysaccharides and Related Probes
Sphingolipids
Structure and Activity
Sphingolipids are essential components of the plasma membrane
of eukaryotic cells, where they are typically found in the outer leaflet. Although particularly abundant in mammalian cells, sphingolipids are also present in Saccharomyces cerevisiae,1 other fungi and
plants. Sphingolipids differ from phospholipids in that they are based
on a lipophilic amino alcohol (sphingosine, Figure 13.3.1) rather than
glycerol. Sphingolipids play important roles in signal transduction
processes2,3 (Chapter 17). Genetic defects in enzymes in the metabolic
pathways of sphingolipid synthesis and degradation, including those
involved in type I Gaucher (Ashkenazi) disease, type A Niemann–
Pick disease, Krabbe disease,4–8 and other lysosomal storage diseases,
can be detected at the cellular level using our fluorescent analogs of
sphingolipids.
Ceramides are the biological building blocks of more complex
sphingolipids. Metabolism of ceramides typically occurs in Golgi and
endoplasmic reticulum membranes, and fluorescent ceramide analogs
(Section 12.4) are important probes for measuring the intracellular distribution and transport of the labeled molecules in live cells.9
If the hydroxyl group of the ceramide is esterified to phosphocholine, the sphingolipid is a sphingomyelin (Figure 13.3.1). The main pathway of sphingomyelin biosynthesis in mammalian cells is based on the
transfer of phosphocholine from glycerophosphocholine to ceramide,
catalyzed by sphingomyelin synthase in the Golgi membrane. Synthesis
is followed by exocytosis of the sphingomyelin to the plasma membrane, apparently via a vesicular pathway and flip-flop to the outer
membrane.2 Sphingomyelinases, which are functionally analogous to
phospholipase C in their chemistry, hydrolyze sphingomyelins back to
ceramides. Generation of ceramides by hydrolysis of sphingomyelins
appears to play a role in mediating the effects of exposure to tumor necrosis factor–α10 (TNF-α), γ-interferon and several other agents, all of
which induce an apoptosis-like cell death.11–15 Section15.5 describes our
extensive selection of reagents for following the diverse morphological
HOCH
+
(CH ) NCH CH O
33
2 2
2
CH
CH
NH
OH
2
O
Figure 13.3.2 BODIPY® FL C12-glucocerebroside.
Figure 13.3.3 BODIPY® FL C5-ganglioside GM1 (B13950).
CH(CH ) CH
2 12 3
Sphingosine
O
P
CH
and biochemical changes that occur during apoptosis. Sensitive fluorescence-based measurements of sphingomyelinase activity using natural,
unlabeled sphingomyelin as the substrate can be carried out using our
Amplex® Red Sphingomyelinase Assay Kit (A12220), described below.
In glycosylsphingolipids, the free hydroxyl group of the ceramide
is glycosylated to give a sphingosyl glycoside (cerebroside, Figure
13.3.2) or a ganglioside (Figure 13.3.3). These glycosphingolipids
form cell-type–specific patterns at the cell surface that change with
cell growth, differentiation, viral transformation and oncogenesis.16
−
O CH
2
CH
CH
NH
OH
C
CH
HO
CH(CH ) CH
2 12
3
OH
O
O O CH 2
HO
R
HOCH
2
CH
CH
NH
OH
C
CH
CH
NH
OH
C
OH
Sphingomyelin
CH
CH(CH ) CH
2 12
3
CH
CH(CH ) CH
2 12
3
O
R
Cerebroside
O
R
Ceramide
Figure 13.3.1 Sphingomyelins, ceramides and cerebrosides are examples of sphingolipids derived from sphingosine. R represents the hydrocarbon tail portion of a fatty acid residue.
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Fluorescent Probes
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566
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IMPORTANT
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manual
are
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or more
Limited
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referonto
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
the Appendix on
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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Chapter 13 — Probes for Lipids and Membranes
Section 13.3 Sphingolipids, Steroids, Lipopolysaccharides and Related Probes
Glycosphingolipids interact at the cell surface with toxins, viruses and bacteria, as well as with receptors and enzymes17 and are involved in cell-type–specific adhesion processes.16 Gangliosides
modulate the trophic factor–stimulated dimerization, tyrosine phosphorylation and subsequent
signal transduction events of several tyrosine kinase receptors.17 Ganglioside GM1 has anti-neurotoxic, neuroprotective and neurorestorative effects on various central neurotransmitter systems.18 Gangliosides, including ganglioside GM1, partition into lipid rafts—detergent-insoluble,
sphingolipid- and cholesterol-rich membrane microdomains that form lateral assemblies in the
plasma membrane.19–25 We offer Vybrant® Lipid Raft Labeling Kits (V34403, V34404, V34405),
as well as Alexa Fluor® dye conjugates of subunit B of cholera toxin (Section 7.7), a protein that
selectively binds to ganglioside GM1 in lipid rafts.
Figure 13.3.4 N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4adiaza-s-indacene-3-pentanoyl)sphingosine (BODIPY® FL
C5-ceramide, D3521).
BODIPY® Sphingolipids
Ceramides (N-acylsphingosines), like diacylglycerols, are lipid second messengers that function in signal transduction processes.26–28 The concentration-dependent spectral properties of
BODIPY® FL C5-ceramide (D3521, B22650; Figure 13.3.4), BODIPY® FL C5-sphingomyelin29–31
(D3522, Figure 13.3.5) and BODIPY® FL C12-sphingomyelin32 (D7711) make them particularly
suitable for investigating sphingolipid transport, metabolism and microdomains, 31,33–37 in addition to their well-documented use as structural markers for the Golgi complex 38 (Section
12.4, Figure 13.3.6). BODIPY® FL C5-ceramide can be visualized by fluorescence microscopy39,40
(Figure 13.3.7, Figure 13.3.8) or by electron microscopy following diaminobenzidine (DAB)
photoconversion to an electron-dense product41 (Fluorescent Probes for Photoconversion of
Diaminobenzidine Reagents—Note 14.2).
Our range of BODIPY® sphingolipids also includes the long-wavelength light–excitable
BODIPY® TR ceramide42,43 (D7540, Figure 13.3.9), as well as BODIPY® FL C5-lactosylceramide44–49
(D13951), BODIPY® FL C5-ganglioside GM150 (B13950, Figure 13.3.3) and BODIPY® FL C12galactocerebroside51 (D7519). All Molecular Probes® sphingolipids are prepared from D-erythrosphingosine and therefore have the same stereochemical conformation as natural biologically
active sphingolipids.52
Complexing fluorescent lipids with bovine serum albumin (BSA) facilitates cell labeling by
eliminating the need for organic solvents to dissolve the lipophilic probe—the BSA-complexed
probe can be directly dissolved in water. We offer four BODIPY® sphingolipid–BSA complexes
for the study of lipid metabolism and trafficking, including BODIPY® FL C5-ceramide, BODIPY®
TR ceramide, BODIPY® FL C5-ganglioside GM1 and BODIPY® FL C5-lactosylceramide, each complexed with defatted BSA (B22650, B34400, B34401, B34402, respectively).
BODIPY® FL C5-ceramide has been used to investigate the linkage of sphingolipid metabolism
to protein secretory pathways53–56 and neuronal growth.47,57 Internalization of BODIPY® FL C5sphingomyelin from the plasma membrane of human skin fibroblasts results in a mixed population of labeled endosomes that can be distinguished based on the concentration-dependent green
Figure 13.3.7 Cells in the notochord rudiment of a zebrafish embryo undergoing mediolateral intercalation to
lengthen the forming notochord. BODIPY® FL C5-ceramide
(D3521) localizes in the interstitial fluid of the zebrafish
embryo and freely diffuses between cells, illuminating
cell boundaries. This confocal image was obtained using a
Bio-Rad® MRC-600 microscope. Image contributed by Mark
Cooper, University of Washington.
Figure 13.3.8 Nucleus and Golgi apparatus of a bovine
pulmonary artery endothelial cell (BPAEC) labeled
with Hoechst 33342 (H1399, H3569, H21492) and
the BSA complex of BODIPY® FL C5-ceramide (D3521,
B22650), respectively.
Figure 13.3.5 N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4adiaza-s-indacene-3-pentanoyl)sphingosyl phosphocholine
(BODIPY® FL C5-sphingomyelin, D3522).
Figure 13.3.6 Selective staining of the Golgi apparatus
using the green-fluorescent BODIPY® FL C5-ceramide
(D3521) (top). At high concentrations, the BODIPY® FL
fluorophore forms excimers that can be visualized using
a red longpass optical filter (bottom). The BODIPY® FL
C5-ceramide accumulation in the trans-Golgi is sufficient
for excimer formation (J Cell Biol (1991) 113:1267). Images
contributed by Richard Pagano, Mayo Foundation.
Figure 13.3.9 BODIPY® TR ceramide (N-((4-(4,4-difluoro5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)phenoxy)
acetyl)sphingosine; D7540).
™
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Molecular
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A Guide
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567
Chapter 13 — Probes for Lipids and Membranes
Section 13.3 Sphingolipids, Steroids, Lipopolysaccharides and Related Probes
(~515 nm) or red (~620 nm) emission of the probe31 (Figure 13.3.6). BODIPY® C5-sphingomyelin
has also been used to assess sphingomyelinase gene transfer and expression in hematopoietic stem
and progenitor cells.4 Studies by Martin and Pagano have shown that the internalization routes
for BODIPY® FL C5-glucocerebroside follow both endocytic and nonendocytic pathways and are
quite different from those for BODIPY® FL C5-sphingomyelin.58
BODIPY® FL C5-lactosylceramide, BODIPY® FL C5-ganglioside GM1 and BODIPY® FL
cerebrosides are useful tools for the study of glycosphingolipid transport and signaling pathways in cells59,60 and for diagnosis of lipid-storage disorders such as Niemann–Pick disease,61
Gaucher disease, GM1 gangliosidosis, Morquio syndrome and type IV mucolipidosis 6,49,62–67 (MLIV). Addition of BODIPY® FL C5-lactosylceramide to the culture medium of cells from patients
with sphingolipid-storage diseases (sphingolipidosis) results in fluorescent product accumulation in lysosomes, whereas this probe accumulates in the Golgi apparatus of normal cells and
cells from patients with other storage diseases.46,48 BODIPY® FL C5-ganglioside GM1 has been
shown to form cholesterol-enhanced clusters in membrane complexes with amyloid β-protein
in a model of Alzheimer disease amyolid fibrils.68 As observed by fluorescence microscopy, the
colocalization of BODIPY® FL C5-ganglioside GM1 and fluorescent cholera toxin B conjugates
(Section 7.7) provides a direct indication of the association of these molecules in lipid rafts50
(Figure 13.3.10).
NBD Sphingolipids
Figure 13.3.10 A J774 mouse macrophage cell sequentially stained with BODIPY® FL ganglioside GM1 (B13950)
and then with Alexa Fluor® 555 dye–labeled cholera toxin
subunit B (C22843, C34776; also available as a component
of V34404). The cell was then treated with an anti–CT-B antibody (a component of V34404) to induce crosslinking. Alexa
Fluor® 555 dye fluorescence (top panel, red) and BODIPY® FL
dye fluorescence (middle panel, green) were imaged separately and overlaid to emphasize the coincident staining
(bottom panel, yellow). Nuclei were stained with blue-fluorescent Hoechst 33342 (H1399, H3570, H21492).
Figure 13.3.11 NBD C6-ceramide (6-((N-(7-nitrobenz-2-oxa1,3-diazol-4-yl)amino)hexanoyl)sphingosine, N1154).
NBD C6 -ceramide (N1154, Figure 13.3.11) and NBD C6 -sphingomyelin (N3524) analogs
predate their BODIPY® counterparts and have been extensively used for following sphingolipid
metabolism in cells9,59,69,70 and in multicellular organisms.71 As with BODIPY® FL C5-ceramide,
we also offer NBD C6 -ceramide complexed with defatted BSA (N22651) to facilitate cell loading
without the use of organic solvents to dissolve the probe. Koval and Pagano have prepared NBD
analogs of both the naturally occurring D-erythro and the nonnatural L-threo stereoisomers of
sphingomyelin and have compared their intracellular transport behavior in Chinese hamster
ovary (CHO) fibroblasts.72
NBD C6 -ceramide lacks the useful concentration-dependent optical properties of the
BODIPY® FL analog and is less photostable; however, the fluorescence of NBD C 6 -ceramide is
apparently sensitive to the cholesterol content of the Golgi apparatus, a phenomenon that is not
observed with BODIPY® FL C5-ceramide. If NBD C6 -ceramide–containing cells are starved for
cholesterol, the NBD C6 -ceramide that accumulates within the Golgi apparatus appears to be
severely photolabile but this NBD photobleaching can be reduced by stimulation of cholesterol
synthesis. Thus, NBD C6 -ceramide may be useful in monitoring the cholesterol content of the
Golgi apparatus in live cells.73
Vybrant® Lipid Raft Labeling Kits
The Vybrant® Lipid Raft Labeling Kits (V34403, V34404, V34405) are designed to provide
convenient, reliable and extremely bright fluorescent labeling of lipid rafts in live cells. Lipid rafts
are detergent-insoluble, sphingolipid- and cholesterol-rich membrane microdomains that form
lateral assemblies in the plasma membrane.19–25 Lipid rafts also sequester glycophosphatidylinositol (GPI)-linked proteins and other signaling proteins and receptors, which may be regulated by
their selective interactions with these membrane microdomains.50,74–78 Lipid rafts play a role in a
variety of cellular processes—including the compartmentalization of cell-signaling events,79–86
the regulation of apoptosis87–89 and the intracellular trafficking of certain membrane proteins
and lipids90–92—as well as in the infectious cycles of several viruses and bacterial pathogens.93–98
Examining the formation and regulation of lipid rafts is a critical step in understanding these
aspects of eukaryotic cell function.
The Vybrant® Lipid Raft Labeling Kits provide the key reagents for fluorescently labeling lipid
rafts in vivo with our bright and extremely photostable Alexa Fluor® dyes (Figure 13.3.10). Live
cells are first labeled with the green-fluorescent Alexa Fluor® 488, orange-fluorescent Alexa Fluor®
555 or red-fluorescent Alexa Fluor® 594 conjugate of cholera toxin subunit B (CT-B). This CT-B
conjugate binds to the pentasaccharide chain of plasma membrane ganglioside GM1, which selectively partitions into lipid rafts.50,99,100 All Molecular Probes® CT-B conjugates are prepared from
recombinant CT-B and are completely free of the toxic subunit A, thus eliminating any concern
for toxicity or ADP-ribosylating activity. An antibody that specifically recognizes CT-B is then
TheMolecular
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and Labeling
Labeling Technologies
Technologies
The
GuidetotoFluorescent
Fluorescent Probes
Probes and
™
568
IMPORTANT
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described
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covered
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Use Label
License(s).
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NOTICE
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described
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arebycovered
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the Appendix on
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Chapter 13 — Probes for Lipids and Membranes
Section 13.3 Sphingolipids, Steroids, Lipopolysaccharides and Related Probes
used to crosslink the CT-B–labeled lipid rafts into distinct patches
on the plasma membrane, which are easily visualized by fluorescence
microscopy.101,102
Each Vybrant® Lipid Raft Labeling Kit contains sufficient reagents
to label 50 live-cell samples in a 2 mL assay, including:
• Recombinant cholera toxin subunit B (CT-B) labeled with the Alexa
Fluor® 488 (in Kit V34403), Alexa Fluor® 555 (in Kit V34404) or
Alexa Fluor® 594 (in Kit V34405) dye
• Anti–cholera toxin subunit B antibody (anti–CT-B)
• Concentrated phosphate-buffered saline (PBS)
• Detailed labeling protocols
Because they are compatible with various multilabeling schemes,
the Vybrant® Lipid Raft Labeling Kits can also serve as important tools
for identifying physiologically significant membrane proteins that associate with lipid rafts. Cells can be labeled with other live-cell probes
during the lipid raft labeling protocol or immediately following the
antibody crosslinking step, depending on the specific labeling requirements of the other probes. Alternatively, once the lipid rafts have been
labeled and crosslinked, the cells can be fixed for long-term storage
or fixed and permeabilized for subsequent labeling with antibodies or
other probes that are impermeant to live cells.
Amplex® Red Sphingomyelinase Assay Kit
The Amplex® Red Sphingomyelinase Assay Kit (A12220) is designed for measuring sphingomyelinase activity in solution using a
fluorescence microplate reader or fluorometer103 (Figure 13.3.12). This
assay should be useful for screening sphingomyelinase activators or
inhibitors or for detecting sphingomyelinase activity in cell and tissue
extracts. The assay, which uses natural sphingomyelin as the principal substrate, employs an enzyme-coupled detection scheme in which
phosphocholine liberated by the action of sphingomyelinase is cleaved
by alkaline phosphatase to generate choline. Choline is, in turn, oxidized by choline oxidase, generating H2O2, which drives the conversion
of the Amplex® Red reagent (A12222, A22177; Section 10.5) to red-fluorescent resorufin. This sensitive assay technique has been employed to
detect activation of acid sphingomyelinase associated with ultraviolet
•
•
•
•
•
•
•
•
•
•
•
Amplex® Red reagent
Dimethylsulfoxide (DMSO)
Horseradish peroxidase (HRP)
H2O2 for use as a positive control
Concentrated reaction buffer
Choline oxidase from Alcaligenes sp.
Alkaline phosphatase from calf intestine
Sphingomyelin
Triton X-100
Sphingomyelinase from Bacillus sp.
Detailed protocols
Each kit provides sufficient reagents for approximately 500 assays
using a fluorescence microplate reader and a reaction volume of 200 µL
per assay.
Steroids
Most steroids are neutral lipids and, as such, localize primarily within the cell’s membranes, in lipid vacuoles and bound to certain lipoproteins. Fluorescent analogs of these biomolecules, most of
which are derived from BODIPY® and NBD dyes, are highly lipophilic
probes. One application of these probes is to detect enzymatic activity—either in vitro or in vivo—through hydrolysis of the fatty acid
esters to fluorescent fatty acids.106 Although the substrates and products in these enzyme assays typically have similar fluorescence properties, they are readily extracted by an organic solvent and separated by
chromatography.
We have also developed sensitive fluorometric assays for cholesterol, cholesteryl esters and enzymes that metabolize natural cholesterol derivatives; the assay reagents and protocols are available in our
Amplex® Red Cholesterol Assay Kit (A12216) described below. A review
of the cellular organization, functions and transport of cholesterol has
recently been published.107
BODIPY® Cholesteryl Esters
6000
Cholesteryl esters consist of a fatty acid esterified to the 3β-hydroxyl
group of cholesterol (Figure 13.3.13). These very nonpolar species are
the predominant lipid components of atherosclerotic plaque and lowand high-density lipoprotein (LDL and HDL) cores. We offer cholesteryl esters of three of our BODIPY® fatty acids—BODIPY® FL C12
(C3927MP), BODIPY® 542/563 C11 (C12680) and BODIPY® 576/589 C11
(C12681)—all of which have long-wavelength visible emission. BODIPY®
5000
Fluorescence
radiation–induced apoptosis104 and to characterize an insecticidal
sphingomyelinase C produced by Bacillus cereus.105
The Amplex® Red Sphingomyelinase Assay Kit contains:
4000
1000
3000
800
600
2000
400
1000
200
0
0
0
10
20
0
0.2
30
0.4
40
0.6
50
Sphingomyelinase (mU/mL)
Figure 13.3.12 Measurement of sphingomyelinase activity using the Amplex® Red Sphingomyelinase Assay Kit (A12220). Each reaction contained 50 µM Amplex® Red reagent, 1 U/mL
horseradish peroxidase (HRP), 0.1 U/mL choline oxidase, 4 U/mL of alkaline phosphatase,
0.25 mM sphingomyelin and the indicated amount of Staphylococcus aureus sphingomyelinase in 1X reaction buffer. Reactions were incubated at 37°C for one hour. Fluorescence was
measured with a fluorescence microplate reader using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm.
Figure 13.3.13 Cholesteryl BODIPY® FL C12 (cholesteryl 4,4-difluoro-5,7-dimethyl-4-bora3a,4a-diaza-s-indacene-3-dodecanoate; C3927MP).
™
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A Guide
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TheMolecular
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Probes®
Handbook:
A Guide
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Probes
and
Labeling
Technologies
IMPORTANT
NOTICE:described
The products
described
this covered
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or moreUse
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UseLicense(s).
Label License(s).
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to the
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IMPORTANT NOTICE
: The products
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bycovered
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Label
Please
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Chapter 13 — Probes for Lipids and Membranes
Section 13.3 Sphingolipids, Steroids, Lipopolysaccharides and Related Probes
FL cholesteryl ester can be used as a tracer of cholesterol transport and
receptor-mediated endocytosis of lipoproteins by fluorescence microscopy108–110 (Figure 13.3.14) and as a general nonexchangeable membrane
marker. Addition of methyl β-cyclodextrin to BODIPY® FL cholesteryl
ester is reported to facilitate its uptake by cells and tissues.111 Researchers
have extensively used BODIPY® FL cholesteryl ester to measure cholesteryl ester–transfer protein (CETP) activity using fluorescence microplate readers.112–115 The longer-wavelength BODIPY® 542/563 and
BODIPY® 576/589 cholesteryl esters likely have similar applications.
Side Chain–Modified Cholesterol Analog
We offer an NBD-labeled cholesterol analog in which the fluorophore replaces the terminal segment of cholesterol’s flexible alkyl
tail. The environment-sensitive NBD fluorophore of the NBD cholesterol analog (N1148) localizes in the membrane’s interior, unlike the
anomalous positioning of NBD-labeled phospholipid acyl chains.116 As
with other NBD lipid analogs, this probe is useful for investigating lipid
transport processes117,118 and lipid–protein interactions.119,120 NBD cholesterol is selectively taken up by high-density lipoproteins via the scavenger receptor B1.117 A lipid droplet–specific protein binds unesterified
NBD cholesterol with extremely high affinity117 (Kd = 2 nM).
Figure 13.3.14 Selective uptake of cholesteryl esters (CE) in rat ovarian granulosa cells as monitored with cholesteryl BODIPY® FL C12 (C3927MP). The hormone-stimulated cells internalized and
stored CEs derived from reconstituted high-density lipoprotein (HDL)–BODIPY® CE complexes (J
Biol Chem (1996) 271:16208). A low-light (<100 µW beam power) computerized imaging system
minimized any photobleaching of the fluorophore. This pseudocolored image uses yellow-green
to illustrate the low-level fluorescence of the cytoplasmic membranes, yellow to illustrate the
medium-level fluorescence of the Golgi, and red to illustrate the high-level fluorescence of the
lipid droplets. Image contributed by Eve Reaven, VA Medical Center, Palo Alto, California.
6000
Fluorescence
5000
Amplex® Red Cholesterol Assay Kit
4000
3000
The Amplex® Red Cholesterol Assay Kit (A12216) provides an exceptionally sensitive assay for both cholesterol and cholesteryl esters
in complex mixtures and is suitable for use with either fluorescence
microplate readers or fluorometers. The assay provided in this kit is
designed to detect as little as 5 ng/mL (5 × 10 –4 mg/dL) cholesterol
(Figure 13.3.15) and to accurately measure the cholesterol or cholesteryl
ester content in the equivalent of 0.01 µL of human serum.121 The assay
uses an enzyme-coupled reaction scheme in which cholesteryl esters
are hydrolyzed by cholesterol esterase into cholesterol, which is then
oxidized by cholesterol oxidase to yield H2O2 and the corresponding
ketone steroidal product (Figure 13.3.16). The H2O2 is then detected
using the Amplex® Red reagent in combination with horseradish peroxidase (HRP).
30
20
2000
10
1000
0
0
0
1
2
0
0.005 0.010 0.015
3
4
Cholesterol (µg/mL)
Figure 13.3.15 Detection of cholesterol using the Amplex® Red Cholesterol Assay Kit
(A12220). Each reaction contained 150 µM Amplex® Red reagent, 1 U/mL horseradish
peroxidase (HRP), 1 U/mL cholesterol oxidase, 1 U/mL cholesterol esterase and the indicated amount of cholesterol in 1X reaction buffer. Reactions were incubated at 37°C for
30 minutes. Fluorescence was measured with a fluorescence microplate reader using excitation at 560 ± 10 nm and fluorescence detection at 590 ± 10 nm. The insert above shows the
high sensitivity and excellent linearity of the assay at low cholesterol levels (0–10 ng/mL).
Š
Figure 13.3.16 Enzyme-coupled Amplex® Red assays. Enzyme reactions that produce H2O2 can be made into Amplex® Red assays. The Amplex® Red Cholesterol Assay Kit (A12216) uses cholesterol oxidase to produce H2O2, which is then detected by the Amplex® Red reagent in the presence of horseradish peroxidase
(HRP). Similarly, the Amplex® Red Acetylcholine/Acetylcholinesterase Assay Kit (A12217) uses choline oxidase to produce H2O2.
The
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The
Molecular
A Guide
Guide to
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Fluorescent Probes
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IMPORTANT
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covered
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one or more
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Chapter 13 — Probes for Lipids and Membranes
Section 13.3 Sphingolipids, Steroids, Lipopolysaccharides and Related Probes
The Amplex® Red cholesterol assay is continuous and requires no
separation or wash steps. These characteristics make the assay particularly well suited for the rapid and direct analysis of cholesterol in blood
and food samples using automated instruments. By performing two
separate measurements in the presence and absence of cholesterol esterase, this assay is also potentially useful for determining the fraction
of cholesterol that is in the form of cholesteryl esters within a sample. In
addition, by adding an excess of cholesterol to the reaction, this assay
can be used to sensitively detect the activity of cholesterol oxidase. The
Amplex® Red Cholesterol Assay Kit contains:
•
•
•
•
•
•
•
•
•
Amplex® Red reagent
Dimethylsulfoxide (DMSO)
Horseradish peroxidase (HRP)
H2O2 for use as a positive control
Concentrated reaction buffer
Cholesterol oxidase from Streptomyces
Cholesterol esterase from Pseudomonas
Cholesterol for preparation of a standard curve
Detailed protocols
Figure 13.3.17 1,2-Dioleoyl-3-(1-pyrenedodecanoyl)-rac-glycerol (D6562).
Fluorescence emission
Each kit provides sufficient reagents for approximately 500 assays
using a fluorescence microplate reader and a reaction volume of 100 µL
per assay.
Fluorescent Triacylglycerol
The fluorescent triacylglycerol 1,2-dioleoyl-3-(1-pyrenedodecanoyl)rac-glycerol (D6562) has a pyrene fatty acid ester replacing one
of the three fatty acyl residues of a natural triacylglycerol (Figure
13.3.17). Pyrene has the important spectral property of forming excimers (Figure 13.3.18) when two fluorophores are in close proximity
during the excited state. Pyrene triacylglycerols are useful for measuring cholesteryl ester transfer protein–mediated triacylglycerol transport
between plasma lipoproteins.122 They are also excellent substrates for
lipoprotein lipase and hepatic triacylglycerol lipase.123
1
2
3
4
350
400
450
500
550
600
Wavelength (nm)
Figure 13.3.18 Excimer formation by pyrene in ethanol. Spectra are normalized to the
371.5 nm peak of the monomer. All spectra are essentially identical below 400 nm after normalization. Spectra are as follows: 1) 2 mM pyrene, purged with argon to remove oxygen; 2)
2 mM pyrene, air-equilibrated; 3) 0.5 mM pyrene (argon-purged); and 4) 2 µM pyrene (argonpurged). The monomer-to-excimer ratio (371.5 nm/470 nm) is dependent on both pyrene
concentration and the excited-state lifetime, which is variable because of quenching by oxygen.
Lipopolysaccharides
Fluorescent Lipopolysaccharides
64
•
•
•
•
•
Alexa Fluor® 488 LPS from E. coli serotype 055:B5 (L23351)
Alexa Fluor® 488 LPS from S. minnesota (L23356)
Alexa Fluor® 568 LPS from E. coli serotype 055:B5 (L23352)
Alexa Fluor® 594 LPS from E. coli serotype 055:B5 (L23353)
BODIPY® FL LPS from E. coli serotype 055:B5 (L23350)
LPS, also known as endotoxins, are a family of complex glycolipid
molecules located on the surface of gram-negative bacteria. LPS play a
large role in protecting the bacterium from host defense mechanisms
and antibiotics. Binding of LPS to the CD14 cell-surface receptor of
phagocytes (Figure 13.3.19) is the key initiation step in the mammalian
immune response to infection by gram-negative bacteria. The structural
Unlabeled cells
Events
We offer fluorescent conjugates of lipopolysaccharides (LPS) from
Escherichia coli and Salmonella minnesota (Section 16.1, Table 16.1),
including:
Labeled cells
0
100
101
102
103
104
Alexa Fluor® 488 LPS fluorescence
Figure 13.3.19 Flow cytometry analysis of blood using an Alexa Fluor® 488 lipopolysaccharide (LPS). Human blood was incubated with Alexa Fluor® 488 LPS from Escherichia coli
(L23351) and anti-CD14 antibody on ice for 20 minutes. The red blood cells were lysed and
the sample was analyzed on a flow cytometer equipped with a 488 nm Ar-Kr excitation
source and a 525 ± 12 nm bandpass emission filter. Monocytes were identified based on their
light scatter and CD14 expression.
™
The
Handbook:
A Guide
to Fluorescent
Probes
and
Labeling
Technologies
TheMolecular
MolecularProbes
Probes®
Handbook:
A Guide
to Fluorescent
Probes
and
Labeling
Technologies
IMPORTANT
NOTICE:described
The products
described
this covered
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by oneLimited
or moreUse
Limited
UseLicense(s).
Label License(s).
to the
Appendix
IMPORTANT NOTICE
: The products
in this
manualin are
bycovered
one or more
Label
PleasePlease
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Appendix
on on
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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571
Chapter 13 — Probes for Lipids and Membranes
OH
O
O
HO
P
OH
O
O
O
O
O
O
NH HO
NH O
O
P
OH
OH
O
O
O
O
O
O
O
HO
O
O
Figure 13.3.20 Structure of the lipid A component of
Salmonella minnesota lipopolysaccharide.
Section 13.3 Sphingolipids, Steroids, Lipopolysaccharides and Related Probes
core of LPS, and the primary determinant of its biological activity, is an N-acetylglucosamine
derivative, lipid A (Figure 13.3.20). Two plasma proteins, LPS-binding protein (LBP) and soluble
CD14 (sCD14), play primary roles in transporting LPS and mediating cellular responses.124–129 If
the fatty acid residues are removed from the lipid A component, the toxicity of the LPS can be reduced significantly; however, the mono- or diphosphoryl forms of lipid A are inherently toxic. In
many gram-negative bacterial infections, LPS are responsible for clinically significant symptoms
like fever, low blood pressure and tissue edema, which can lead to disseminated intravascular
coagulation, organ failure and death. Studies also clearly indicate that LPS induce various signal transduction pathways, including those involving protein kinase C130,131 and protein myristylation,132 and stimulate a variety of immunochemical responses, including B lymphocyte133
and G-protein activation.134
The fluorescent BODIPY® FL and Alexa Fluor® LPS conjugates, which are labeled with succinimidyl esters of these dyes, allow researchers to follow LPS binding, transport and cell internalization processes. Lipopolysaccharide internalization activates endotoxin-dependent signal
transduction in cardiomyocytes.135 The Alexa Fluor® 488 LPS conjugates (L23351, L23356) selectively label microglia in a mixed culture containing oligodendrocyte precursors, astrocytes
and microglia.136 A biologically active conjugate of galactose oxidase–oxidized S. minnesota LPS
and our Alexa Fluor® 488 hydrazide (A10436, Section 3.3; A10440) has been used to elucidate
molecular mechanisms of septic shock.137
The BODIPY® FL derivative of LPS from E. coli strain LCD25 (L23350) was used to measure
the transfer rate of LPS from monocytes to high-density lipoprotein138 (HDL). Another study
utilized a BODIPY® FL derivative of LPS from S. minnesota to demonstrate transport to the Golgi
apparatus in neutrophils,124,125 although this could have been due to probe metabolism. It has
been reported that organelles other than the Golgi are labeled by some fluorescent or nonfluorescent LPS.139,140 Cationic lipids are reported to assist the translocation of fluorescent lipopolysaccharides into live cells;141 cell surface–bound LPS can be quenched by trypan blue.138 Molecular
Probes® fluorescent LPS can potentially be combined with other fluorescent indicators, such as
Ca 2+-, pH- or organelle-specific stains, for monitoring intracellular localization and real-time
changes in cellular response to LPS.
Pro-Q® Emerald 300 Lipopolysaccharide Gel Stain Kit
Fluorescence
Fluorescence
Figure 13.3.21 Lipopolysaccharide staining with the
Pro-Q® Emerald 300 Lipopolysaccharide Gel Stain Kit. Lipopolysaccharides (LPS) were electrophoresed through a 13%
acrylamide gel and stained using the Pro-Q® Emerald 300
Lipopolysaccharide Gel Stain Kit (P20495). From left to right,
the lanes contain: CandyCane™ glycoprotein molecular
weight standards (~250 ng/band), blank, 4, 1 and 0.25 µg
of LPS from Escherichia coli smooth serotype 055:B5 and
4, 1 and 0.25 µg of LPS from E. coli rough mutant EH100
(Ra mutant).
The Pro-Q® Emerald 300 Lipopolysaccharide Gel Stain Kit (P20495) provides a simple, rapid
and highly sensitive method for staining lipopolysaccharides (LPS) in gels (Figure 13.3.21, Figure
13.3.22, Figure 13.3.23). The structure of this important class of molecules can be analyzed by
SDS-polyacrylamide gel electrophoresis, during which the heterogeneous mixture of polymers
separates into a characteristic ladder pattern. This ladder has conventionally been detected using
silver staining.142–144 However, despite the long and complex procedures required, silver staining
provides poor sensitivity and cannot differentiate LPS from proteins in the sample. An alternative
0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
Figure 13.3.22 Characterization of lipopolysaccharides.
Lipopolysaccharides (LPS) from Escherichia coli smooth
serotype 055:B5 were loaded onto a 13% polyacrylamide
gel. Following electrophoresis, the gel was stained using
the Pro-Q® Emerald 300 Lipopolysaccharide Gel Stain Kit
(P20495), and the fluorescence was measured for the lane. A
plot of fluorescence signal versus the relative distance from
the dye front shows a characteristic laddering profile for
smooth-type LPS.
™
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2
3
4
Figure 13.3.23 Linearity of the Pro-Q® Emerald 300 stain for
lipopolysaccharide (LPS) detection. A dilution series of lipopolysaccharides from Escherichia coli smooth serotype 055:B5
was loaded onto a 13% polyacrylamide gel. Following electrophoresis, the gel was stained using the Pro-Q® Emerald
300 Lipopolysaccharide Gel Stain Kit (P20495) and the same
band from each lane was quantitated using a CCD camera. A
plot of the fluorescence intensity versus the mass of LPS
loaded shows a linear range over two orders of magnitude.
The
MolecularProbes®
Probes Handbook:
Handbook: AAGuide
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LabelingTechnologies
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572
1
Mass of LPS (µg)
Migration (Rf)
the Appendix on
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Chapter 13 — Probes for Lipids and Membranes
Section 13.3 Sphingolipids, Steroids, Lipopolysaccharides and Related Probes
staining method that makes use of the reaction of the carbohydrates
with detectable hydrazides obtains higher sensitivity, but requires blotting to a membrane and time- and labor-intensive procedures.145–149
By comparison, the staining technology used in the Pro-Q® Emerald
300 Lipopolysaccharide Gel Stain Kit vastly simplifies detection of LPS
in SDS-polyacrylamide gels. The key to this novel methodology is our
bright green-fluorescent Pro-Q® Emerald 300 dye, which covalently
binds to periodate-oxidized carbohydrates of LPS. This dye allows the
detection of as little as 200 pg of LPS in just a few hours using a simple
UV transilluminator. The sensitivity is at least 50–100 times that of silver
staining and requires much less hands-on time. This dye is also used
in our Pro-Q® Emerald 300 and Multiplexed Proteomics® Glycoprotein
Stain Kits (P21855, P21857, M33307; Section 9.4) and may be useful for
detection of other molecules containing carbohydrates or aldehydes.
The Pro-Q® Emerald 300 Lipopolysaccharide Gel Stain Kit contains:
•
•
•
•
•
Pro-Q® Emerald 300 reagent
Pro-Q® Emerald 300 staining buffer
Oxidizing reagent (periodic acid)
Smooth LPS standard from Escherichia coli serotype 055-B5
Detailed protocols
Sufficient materials are supplied to stain ten 8 cm × 10 cm gels,
0.5–0.75 mm thick.
REFERENCES
1. Annu Rev Biochem (1998) 67:27; 2. Prog Lipid Res (1997) 36:153; 3. Biochim Biophys
Acta (1998) 1436:233; 4. Blood (1999) 93:80; 5. Biochim Biophys Acta (1984) 793:169;
6. Clin Chim Acta (1982) 124:123; 7. Clin Chim Acta (1984) 142:313; 8. J Cell Biol (1990)
111:429; 9. Methods Cell Biol (1993) 38:221; 10. J Biol Chem (1993) 268:17762; 11. Cell
Signal (1998) 10:685; 12. Trends Biochem Sci (1995) 20:73; 13. Curr Opin Oncol (1998)
10:552; 14. J Inherit Metab Dis (1998) 21:472; 15. Science (1993) 259:1769; 16. Ann N Y
Acad Sci (1998) 845:139; 17. Ann N Y Acad Sci (1998) 845:57; 18. J Neurochem (1998)
70:1335; 19. J Cell Biol (2003) 162:365; 20. J Lipid Res (2003) 44:655; 21. Eur J Biochem
(2002) 269:737; 22. Science (2000) 290:1721; 23. Mol Membr Biol (1999) 16:145; 24. Trends
Cell Biol (1999) 9:87; 25. Annu Rev Cell Dev Biol (1998) 14:111; 26. Biochemistry (2001)
40:4893; 27. Trends Cell Biol (2000) 10:408; 28. J Biol Chem (1994) 269:3125; 29. Chem
Phys Lipids (1999) 102:55; 30. Ann N Y Acad Sci (1998) 845:152; 31. Biophys J (1997)
72:37; 32. J Cell Biol (1998) 140:39; 33. Histochem Cell Biol (2008) 130:819; 34. Methods
Enzymol (2000) 312:293; 35. Methods Enzymol (2000) 312:523; 36. Methods (2005)
36:186; 37. J Cell Biol (1996) 134:1031; 38. J Cell Biol (1991) 113:1267; 39. Cytometry (1993)
14:251; 40. J Cell Biol (1993) 120:399; 41. Eur J Cell Biol (1992) 58:214; 42. Mol Biochem
Parasitol (2000) 106:21; 43. Infect Immun (2000) 68:5960; 44. J Cell Biol (2001) 154:535;
45. Am J Physiol Lung Cell Mol Physiol (2001) 280:L938; 46. Nat Cell Biol (1999) 1:386;
47. J Neurochem (1999) 73:1375; 48. Lancet (1999) 354:901; 49. Proc Natl Acad Sci U S A
(1998) 95:6373; 50. J Cell Biol (1999) 147:447; 51. J Cell Biol (2002) 157:327; 52. Biophys
J (1999) 77:1498; 53. J Cell Sci (2006)119:2084; 54. Mol Biol Cell (1995) 6:135; 55. J Biol
Chem (1993) 268:4577; 56. Biochemistry (1992) 31:3581; 57. J Biol Chem (1993) 268:14476;
58. J Cell Biol (1994) 125:769; 59. Biochim Biophys Acta (1992) 1113:277; 60. Brain Res
(1992) 597:108; 61. Anal Biochem (2001) 293:204; 62. Biochim Biophys Acta (1999)
1455:85; 63. Traffic (2000) 1:807; 64. J Biol Chem (1993) 268:14861; 65. Biochim Biophys
Acta (1987) 915:87; 66. Biochem Biophys Res Comm (1965) 18:221; 67. Anal Biochem
(1984) 136:223; 68. J Biol Chem (2001) 276:24985; 69. Adv Cell Mol Biol Membranes
(1993) 1:199; 70. Biochim Biophys Acta (1991) 1082:113; 71. Parasitology (1992) 105:81;
72. J Cell Biol (1989) 108:2169; 73. Proc Natl Acad Sci U S A (1993) 90:2661; 74. Proc Natl
Acad Sci U S A (2003) 100:5813; 75. J Immunol (2003) 170:1329; 76. J Membr Biol (2002)
189:35; 77. Proc Natl Acad Sci U S A (2001) 98:9098; 78. Mol Biol Cell (1999) 10:3187;
79. Biochim Biophys Acta (2003) 1610:247; 80. Annu Rev Immunol (2003) 21:457; 81. Mol
Immunol (2002) 38:1247; 82. Nat Rev Immunol (2002) 2:96; 83. Biol Res (2002) 35:127;
84. Nat Rev Mol Cell Biol (2000) 1:31; 85. J Exp Med (1999) 190:1549; 86. J Cell Biol (1998)
143:637; 87. Immunity (2003) 18:655; 88. J Biol Chem (2002) 277:39541; 89. Biochem
Biophys Res Commun (2002) 297:876; 90. Biol Chem (2002) 383:1475; 91. J Cell Biol
(2001) 153:529; 92. J Cell Sci (2001) 114:3957; 93. J Virol (2003) 77:9542; 94. Exp Cell Res
(2003) 287:67; 95. Traffic (2002) 3:705; 96. J Clin Virol (2001) 22:217; 97. Curr Biol (2000)
10:R823; 98. J Virol (2000) 74:3264; 99. Biochemistry (1996) 35:16069; 100. Mol Microbiol
(1994) 13:745; 101. J Cell Biol (1998) 141:929; 102. J Biol Chem (1994) 269:30745; 103. Am
J Pathol (2002) 161:1061; 104. J Biol Chem (2001) 276:11775; 105. Eur J Biochem (2004)
271:601; 106. J Lipid Res (1995) 36:1602; 107. Nat Rev Mol Cell Biol (2008) 9:125; 108. Proc
Natl Acad Sci U S A (2001) 98:1613; 109. J Biol Chem (1997) 272:25283; 110. PLoS ONE
(2007) 2:e511; 111. Am J Physiol (1999) 277:G1017; 112. Biochemistry (1995) 34:12560;
113. Chem Phys Lipids (1995) 77:51; 114. Lipids (1994) 29:811; 115. J Lipid Res (1993)
34:1625; 116. J Phys Chem (1999) 103:8180; 117. J Biol Chem (2000) 275:12769; 118. J Lipid
Res (1999) 40:1747; 119. Biochim Biophys Acta (1999) 1437:37; 120. J Biol Chem (1999)
274:35425; 121. J Biochem Biophys Methods (1999) 38:43; 122. J Biochem (Tokyo)
(1998) 124:237; 123. Lipids (1988) 23:605; 124. J Exp Med (1999) 190:523; 125. J Exp
Med (1999) 190:509; 126. J Biol Chem (1996) 271:4100; 127. J Exp Med (1995) 181:1743;
128. J Exp Med (1994) 180:1025; 129. J Exp Med (1994) 179:269; 130. J Biol Chem (1984)
259:10048; 131. J Exp Med (1996) 183:1899; 132. Proc Natl Acad Sci U S A (1986) 83:5817;
133. Adv Immunol (1979) 28:293; 134. Eur J Immunol (1989) 19:125; 135. Circ Res
(2001) 88:491; 136. J Neurosci (2002) 22:2478; 137. Cytometry (2000) 41:316; 138. J Biol
Chem (1999) 274:34116; 139. Electron Microsc Rev (1992) 5:381; 140. J Periodontol
(1985) 56:553; 141. Biotechniques (2000) 28:510; 142. J Clin Microbiol (1990) 28:2627;
143. Microbiol Immunol (1991) 35:331; 144. J Biochem Biophys Methods (1993) 26:81;
145. Electrophoresis (1998) 19:2398; 146. Appl Environ Microbiol (1995) 61:2845;
147. Electrophoresis (1999) 20:462; 148. Electrophoresis (2000) 21:526; 149. Anal Biochem
(1990) 188:285.
DATA TABLE 13.3 SPHINGOLIPIDS, STEROIDS, LIPOPOLYSACCHARIDES AND RELATED PROBES
Cat. No.
B13950
B22650
B34400
B34401
B34402
C3927MP
C12680
C12681
D3521
D3522
D6562
D7519
D7540
MW
1582.50
~66,000
~66,000
~66,000
~66,000
786.98
851.02
809.97
601.63
766.75
1003.54
861.96
705.71
Storage
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
FF,D,L
FF,D,L
FF,D,L,A
FF,D,L
FF,D,L
Soluble
DMSO, EtOH
H2O
H2O
H2O
H2O
CHCl3
CHCl3
CHCl3
CHCl3, DMSO
see Notes
CHCl3
DMSO, EtOH
CHCl3, DMSO
Abs
505
505
589
505
505
505
543
579
505
505
341
505
589
EC
80,000
91,000
65,000
80,000
80,000
86,000
57,000
98,000
91,000
77,000
40,000
85,000
65,000
Em
512
511
616
512
511
511
563
590
511
512
376
511
616
Solvent
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
Notes
1
1, 2
2
1, 2
1, 2
3
3
3
1
1, 4
5, 6
1
continued on next page
™
The
Handbook:
A Guide
to Fluorescent
Probes
and
Labeling
Technologies
TheMolecular
MolecularProbes
Probes®
Handbook:
A Guide
to Fluorescent
Probes
and
Labeling
Technologies
IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on
IMPORTANT NOTICE
: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
www.invitrogen.com/probes
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573
Chapter 13 — Probes for Lipids and Membranes
Section 13.3 Sphingolipids, Steroids, Lipopolysaccharides and Related Probes
DATA TABLE 13.3 SPHINGOLIPIDS, STEROIDS, LIPOPOLYSACCHARIDES AND RELATED PROBES—continued
Cat. No.
MW
Storage
Soluble
Abs
EC
Em
Solvent
Notes
D7711
864.94
FF,D,L
DMSO
505
75,000
513
MeOH
1, 7
D13951
925.91
FF,D,L
DMSO, EtOH
505
80,000
511
MeOH
1
469
21,000
537
MeOH
8
N1148
494.63
L
CHCl3, MeCN
466
22,000
536
MeOH
8
N1154
575.75
FF,D,L
CHCl3, DMSO
N3524
740.88
FF,D,L
see Notes
466
22,000
536
MeOH
4, 8
466
22,000
536
MeOH
2, 8
N22651
~66,000
F,D,L
H2O
For definitions of the contents of this data table, see “Using The Molecular Probes® Handbook” in the introductory pages.
Notes
1. Em for BODIPY® FL sphingolipids shifts to ~620 nm when high concentrations of the probe (>5 mol %) are incorporated in lipid mixtures. (J Cell Biol (1991) 113:1267)
2. This product is a lipid complexed with bovine serum albumin (BSA). Spectroscopic data are for the free lipid in MeOH.
3. The absorption and fluorescence spectra of BODIPY® derivatives are relatively insensitive to the solvent.
4. Chloroform is the most generally useful solvent for preparing stock solutions of phospholipids (including sphingomyelins). Glycerophosphocholines are usually freely soluble in ethanol. Most
other glycerophospholipids (phosphoethanolamines, phosphatidic acids and phosphoglycerols) are less soluble in ethanol, but solutions up to 1–2 mg/mL should be obtainable, using sonication to aid dispersion if necessary. Labeling of cells with fluorescent phospholipids can be enhanced by addition of cyclodextrins during incubation. (J Biol Chem (1999) 274:35359)
5. Alkylpyrene fluorescence lifetimes are up to 110 nanoseconds and are very sensitive to oxygen.
6. Pyrene derivatives exhibit structured spectra. The absorption maximum is usually about 340 nm with a subsidiary peak at about 325 nm. There are also strong absorption peaks below
300 nm. The emission maximum is usually about 376 nm with a subsidiary peak at 396 nm. Excimer emission at about 470 nm may be observed at high concentrations.
7. This product is supplied as a ready-made solution in the solvent indicated under "Soluble."
8. Fluorescence of NBD and its derivatives in water is relatively weak. QY and τ increase and Em decreases in aprotic solvents and other nonpolar environments relative to water. (Biochemistry
(1977) 16:5150, Photochem Photobiol (1991) 54:361)
PRODUCT LIST 13.3 SPHINGOLIPIDS, STEROIDS, LIPOPOLYSACCHARIDES AND RELATED PROBES
Cat. No.
Product
A12216
Amplex® Red Cholesterol Assay Kit *500 assays*
Quantity
A12220
Amplex® Red Sphingomyelinase Assay Kit *500 assays*
B22650
BODIPY® FL C5-ceramide complexed to BSA
5 mg
1 kit
1 kit
B13950
BODIPY® FL C5-ganglioside GM1
25 µg
B34401
BODIPY® FL C5-ganglioside GM1 complexed to BSA
1 mg
B34402
BODIPY® FL C5-lactosylceramide complexed to BSA
D7540
BODIPY® TR ceramide (N-((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)phenoxy)acetyl)sphingosine)
B34400
BODIPY® TR ceramide complexed to BSA
5 mg
C12680
cholesteryl BODIPY® 542/563 C11 (cholesteryl 4,4-difluoro-5-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoate)
1 mg
C12681
cholesteryl BODIPY® 576/589 C11 (cholesteryl 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoate)
1 mg
C3927MP
cholesteryl BODIPY® FL C12 (cholesteryl 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoate)
1 mg
D7519
N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)sphingosyl 1-β-D-galactopyranoside (BODIPY® FL C12-galactocerebroside)
25 µg
D7711
N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)sphingosyl phosphocholine (BODIPY® FL C12-sphingomyelin) *1 mg/mL in DMSO*
250 µL
D3521
N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)sphingosine (BODIPY® FL C5-ceramide)
250 µg
1 mg
250 µg
D13951
N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)sphingosyl 1-β-D-lactoside (BODIPY® FL C5-lactosylceramide)
25 µg
D3522
N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)sphingosyl phosphocholine (BODIPY® FL C5-sphingomyelin)
250 µg
D6562
1,2-dioleoyl-3-(1-pyrenedodecanoyl)-rac-glycerol
L23351
lipopolysaccharides from Escherichia coli serotype 055:B5, Alexa Fluor® 488 conjugate
100 µg
L23352
lipopolysaccharides from Escherichia coli serotype 055:B5, Alexa Fluor® 568 conjugate
100 µg
L23353
lipopolysaccharides from Escherichia coli serotype 055:B5, Alexa Fluor® 594 conjugate
100 µg
L23350
lipopolysaccharides from Escherichia coli serotype 055:B5, BODIPY® FL conjugate
100 µg
L23356
lipopolysaccharides from Salmonella minnesota, Alexa Fluor® 488 conjugate
100 µg
N1154
NBD C6-ceramide (6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)sphingosine)
1 mg
N22651
NBD C6-ceramide complexed to BSA
5 mg
N3524
NBD C6-sphingomyelin (6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)sphingosyl phosphocholine)
N1148
NBD cholesterol (22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol)
P20495
Pro-Q® Emerald 300 Lipopolysaccharide Gel Stain Kit *10 minigels*
1 kit
V34403
Vybrant® Alexa Fluor® 488 Lipid Raft Labeling Kit *50 labelings*
1 kit
V34404
Vybrant® Alexa Fluor® 555 Lipid Raft Labeling Kit *50 labelings*
1 kit
V34405
Vybrant® Alexa Fluor® 594 Lipid Raft Labeling Kit *50 labelings*
1 kit
The
MolecularProbes®
Probes Handbook:
Handbook: AAGuide
Probes and
and Labeling
LabelingTechnologies
Technologies
The
Molecular
Guideto
toFluorescent
Fluorescent Probes
™
574
IMPORTANT
NOTICE:
The products
described
in this manual
coveredare
by covered
one or more
Limited
Use Label
License(s).
Please
refer to thePlease
Appendix
onto
IMPORTANT
NOTICE
: The products
described
in thisaremanual
by one
or more
Limited
Use Label
License(s).
refer
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
the Appendix on
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
www.invitrogen.com/probes
thermofisher.com/probes
1 mg
1 mg
10 mg
Chapter 13 — Probes for Lipids and Membranes
Section 13.4 Dialkylcarbocyanine and Dialkylaminostyryl Probes
13.4 Dialkylcarbocyanine and Dialkylaminostyryl Probes
The dyes in this section are all amphiphilic probes—molecules that comprise a charged
fluorophore that localizes the probe at the membrane’s surface and lipophilic aliphatic "tails"
that insert into the membrane and thus anchor the probe to the membrane. In addition to labeling model membranes, most of these probes are very useful for cell tracing applications (Section
14.4). Table 14.3 lists all of our lipophilic carbocyanine and aminostyryl tracers and compares
their properties and uses. Our FM® dyes, which are also amphiphilic styryl dyes but with less
lipophilic character than the dyes in this section, are particularly useful for labeling membranes
of live cells and for following synaptosome recycling (Section 16.1).
Figure 13.4.3 DiIC12(3) (1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; D383)
Dialkylcarbocyanine Probes
Carbocyanines are among the most strongly light-absorbing dyes known and have proven
to be useful tools in several different areas of research. Carbocyanines with short alkyl tails
attached to the imine nitrogens are employed both as membrane-potential sensors (Section
22.3) and as organelle stains for mitochondria and the endoplasmic reticulum (Section 12.2,
Section 12.4). Those with longer alkyl tails (≥12 carbons) have an overall lipophilic character
that makes them useful for neuronal tracing1 and long-term labeling of cells in culture2,3 (Section
14.4), as well as for noncovalent labeling of lipoproteins (Section 16.1). This section describes the
use and properties of dialkylcarbocyanines as general-purpose probes of membrane structure
and dynamics.
Figure 13.4.4 'DiD'; DiIC18(5) (1,1’-dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine perchlorate; D307).
DiI, DiO, DiD, DiR and Analogs
The most widely used carbocyanine membrane probes have been the octadecyl (C18) indocarbocyanines (D282, D3911; Figure 13.4.1) and oxacarbocyanines (D275, Figure 13.4.2) often
referred to by the generic acronyms DiI and DiO, or more specifically as DiIC18(3) and DiOC18(3),
where the subscript is the number of carbon atoms in each alkyl tail and the bracketed numeral is
the number of carbon atoms in the bridge between the indoline or benzoxazole ring systems. We
also offer several variations on these basic structures (Section 14.4, Table 14.3):
Figure 13.4.5 'DiR'; DiIC18(7) (1,1’-dioctadecyl-3,3,3’,3’-tetramethylindotricarbocyanine iodide; D12731)
• DiI and DiO analogs with unsaturated alkyl tails (Δ9-DiI, D3886; FAST DiO™, D3898; FAST
DiI™, D3899, D7756)
• DiI and DiO analogs with shorter alkyl tails (DiIC12(3), D383; Figure 13.4.3; DiIC16(3), D384;
DiOC16(3), D1125)
• Long-wavelength light–excitable carbocyanines (DiD, D307, D7757; Figure 13.4.4)
• Infrared light–excitable carbocyanine (DiR, D12731; Figure 13.4.5)
• Chloromethylbenzamido DiI and sulfonated DiI and DiO derivatives
Spectral Properties of Dialkylcarbocyanines
The spectral properties of dialkylcarbocyanines are largely independent of the lengths of the
alkyl chains, and are instead determined by the heteroatoms in the terminal ring systems and the
length of the connecting bridge. The DiICn(3) probes have absorption and fluorescence spectra
compatible with rhodamine (TRITC) optical filter sets (Figure 13.4.6), whereas DiOCn(3) analogs
can be used with fluorescein (FITC) optical filter sets (Figure 13.4.7). The emission maxima of
Figure 13.4.1 'DiI'; DiIC18(3) (1,1'-dioctadecyl-3,3,3',3'tetramethylindocarbocyanine perchlorate; D282).
Figure 13.4.2 'DiO'; DiOC18(3) (3,3'-dioctadecyloxacarbocyanine perchlorate; D275).
Figure 13.4.6 Absorption and fluorescence emission spectra
of DiIC18(3) ("DiI") bound to phospholipid bilayer membranes.
Figure 13.4.7 Absorption and fluorescence emission spectra
of DiOC18(3) ("DiO") bound to phospholipid bilayer membranes.
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Chapter 13 — Probes for Lipids and Membranes
Dil
DiD
DiR
Fluorescence emission
DiO
500
600
700
800
900
Wavelength (nm)
Figure 13.4.8 Normalized fluorescence emission spectra of
DiO (D275), DiI (D282), DiD (D307) and DiR (D12731) bound
to phospholipid bilayer membranes.
Section 13.4 Dialkylcarbocyanine and Dialkylaminostyryl Probes
DiIC18(3) and DiOC18(3) incorporated in dioctadecenoylphosphocholine (dioleoyl PC or DOPC)
liposomes (Figure 13.4.8) are similar to those of the dyes in methanol.
The very large molar extinction coefficients of carbocyanine fluorophores are their most outstanding spectral property. Their fluorescence quantum yields are only modest—about 0.07 for
DiI in methanol and about three-times greater in amphiphilic solvents such as octanol.4,5 Their
fluorescence in water is quite weak.6 The excited-state lifetimes of carbocyanine fluorophores in
lipid environments are short (~1 nanosecond), which is an advantage for flow cytometry applications because it allows more excitation/de-excitation cycles during flow transit; the overall decay
is multi-exponential.7 Dialkylcarbocyanines are also exceptionally photostable.8
The red He-Ne laser–excitable indodicarbocyanines such as DiD (DiIC18(5); D307, D7757)
have long-wavelength absorption and red emission (Figure 13.4.8, Figure 13.4.9). Their extinction coefficients are somewhat larger and fluorescence quantum yields much larger than those
of carbocyanines such as DiI.5 Moreover, photoexcitation of DiD seems to cause less collateral
damage than photoexcitation of DiI in live cells.9 The DiIC18(7) tricarbocyanine probe (DiR,
D12731) has excitation and emission in the infrared (Figure 13.4.10), which may make the dye
useful as an in vivo tracer for labeled cells and liposomes in live organisms.4,10
Substituted DiI and DiO Derivatives
We have synthesized various derivatives of DiI, DiO and DiD. All of these derivatives have
octadecyl (C18) tails identical to those of DiI (D282, D3911) and DiO (D275), thereby preserving
the excellent membrane retention characteristics of the parent molecules. A variety of substitutions have been made on the indoline or benzoxazole ring systems:
Figure 13.4.9 Absorption and fluorescence emission spectra
of DiIC18(5) ("DiD") bound to phospholipid bilayer membranes.
• Chloromethylbenzamido DiI derivatives (CellTracker™ CM-DiI; C7000, C7001; Figure 13.4.11)
• Anionic sulfophenyl derivatives11 of DiI and DiO (SP-DiIC18(3), D7777, Figure 13.4.12; SPDiOC18(3), D7778, Figure 13.4.13)
• Sulfonate derivatives of DiI and DiD (DiIC18(3)-DS, D7776, Figure 13.4.14; DiIC18(5)-DS,
D12730, Figure 13.4.15)
Although these derivatives have primarily been developed to provide improved fixation and
labeling in long-term cell tracing applications (Section 14.4), they also offer several features that
can potentially be exploited for investigating membrane structure and dynamics. For researchers wishing to carry out comparative evaluations, our Lipophilic Tracer Sampler Kit (L7781)
provides 1 mg samples of each of nine different carbocyanine derivatives, including several of
the newer substituted derivatives:
• DiI (DiIC18(3))
• DiD (DiIC18(5))
• DiR (DiIC18(7))
Figure 13.4.10 Fluorescence excitation and emission spectra
of DiIC18(7) ("DiR") bound to phospholipid bilayer membranes.
• DiO (DiOC18(3))
• DiA (4-Di-16-ASP)
• DiIC18(3)-DS
The fluorescence quantum yields of the sulfophenyl and phenyl derivatives (measured in methanol) are generally 2- to 3-fold greater than those of DiI and DiO. In particular, we have found that
the sulfophenyl derivatives (SP-DiIC18(3), D7777; SP-DiOC18(3), D7778) bound to phospholipid
model membranes have approximately 5-fold higher quantum yields than DiI and DiO. DiIC18(5)DS (D12730) has been used in combination with an NBD-labeled glycerophosphoserine probe
in a novel resonance energy transfer assay that detects inner monolayer membrane hemifusion,
avoiding erroneous indications of membrane fusion due to lipid mixing and other environmental
effects in the outer monolayer.12 The negative charge and greater water solubility of the sulfonated
carbocyanines results in modified lateral and transverse distributions of these probes in lipid bilayers relative to those of DiI and DiO. This characteristic has been exploited to identify plasma membrane lipid domains that are responsive to electrical stimulation of outer hair cells in the inner ear.13
Figure 13.4.11 CellTracker™ CM-DiI (C7000).
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576
• SP-DiIC18(3)
• SP-DiOC18(3)
• 5,5ʹ-Ph2-DiIC18(3)
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Chapter 13 — Probes for Lipids and Membranes
Section 13.4 Dialkylcarbocyanine and Dialkylaminostyryl Probes
DiI and DiO as Probes of Membrane Structure
The orientation of DiIC18(3) in membranes has been determined by
fluorescence polarization microscopy.14 The long axis of the fluorophore
is parallel to the membrane surface, and the two alkyl chains protrude
perpendicularly into the lipid interior. There are conflicting reports in
the literature regarding the ease of transbilayer migration ("flip-flop")
of lipophilic indocarbocyanines.15–17 The lateral partitioning behavior of
dialkylindocarbocyanines in membranes has been investigated by fluorescence recovery after photobleaching (FRAP),18 calorimetry,19 lifetime
measurements8 and fluorescence resonance energy transfer techniques20
(Fluorescence Resonance Energy Transfer (FRET)—Note 1.2). These
studies demonstrate that the probe distribution between coexisting fluid
and gel phases depends on the similarity of the alkyl chain lengths of the
probe and the lipid. In general, the more dissimilar the lengths, the greater the preference for fluid-phase over gel-phase lipids. For example, the
shorter-chain DiIC12(3) has a substantial preference for the fluid phase
(~6:1) in DOPC, whereas DiIC18(3) is predominantly distributed in the
gel phase21 (~1:10). Consequently, long-chain dialkylcarbocyanines are
among the best probes for detecting particularly rigid gel phases.
Lipophilic carbocyanines have been used to visualize membrane
fusion and cell permeabilization that occurs in response to electric fields,22–24 as well as fusion of liposomes with planar bilayers.25
Membrane fusion can also be measured by fluorescence resonance
energy transfer to DiIC18(3) from dansyl- or NBD-labeled phospholipid donors26 or by direct imaging.27 In Langmuir–Blodgett films,
excited-state energy transfer from DiIC18(3) to DiIC18(5) is exceptionally efficient because of the favorable orientations of the fluorophores.28
Energy transfer from DiIC18(5) to DiIC18(7) should be similarly effective. Lipophilic carbocyanines have also been used to elicit photosensitized destabilization of liposomes, 29 to sensitize photoaffinity labeling
of the viral glycoprotein hemagglutinin, 30 to image membrane domains
in lipid monolayers31 and to develop a fiber-optic potassium sensor.32
DiI and DiO as Probes of Membrane Dynamics
Despite their reasonably good photostability, dialkylcarbocyanines
are widely employed to measure lateral diffusion processes using fluorescence recovery after photobleaching (FRAP) techniques.33–36 Their
lateral diffusion coefficients in isolated fluid- and gel-phase bilayers are
independent of the carbocyanine alkyl chain length.18 Phase-separated
populations of lipophilic carbocyanine dyes can be distinguished by
their diffusion rates and can therefore be used to define lateral domains
in cell membranes.37,38 Combined lateral diffusion measurements of labeled proteins and lipids have demonstrated that transformed39 and
permeabilized40 cells show marked changes in protein diffusion, whereas lipid diffusion rates remain unchanged. In other cases, coupling of
lipid and protein mobility has been identified in the form of relatively
immobilized lipid domains in yeast plasma membranes41 and around
IgE receptor complexes.42 A different photobleaching technique, which
depends on the absence of diffusional fluorescence recovery, was employed to determine lipid flow direction in locomoting cells by following the movement of a photobleached stripe of DiIC16(3)43 (D384).
H3C
The lipophilic aminostyryl probes 4-Di-10-ASP (D291, Figure
13.4.16), DiA (4-Di-16-ASP, D3883; Figure 13.4.17) and FAST DiA™
(D7758, Figure 13.4.18) insert in membranes with their two alkyl
CH
N
CH CH
CH3
N
(CH2)��
(CH2)��
O3�
�O3H
CH3
CH3
Figure 13.4.12 1,1’-Dioctadecyl-6,6’-di(4-sulfophenyl)-3,3,3’,3’-tetramethylindocarbocyanine
(SP-DiIC18(3), D7777).
O
CH
N
CH CH
(CH2)��
O3�
N
(CH2)��
N�
CH3
O
�O3
CH3
Figure 13.4.13 3,3’-Dioctadecyl-5,5’-di(4-sulfophenyl)oxacarbocyanine, sodium salt
(SP-DiOC18(3), D7778).
H3C
O3�
H3C
CH3
CH
N
CH CH
CH3
�O3H
N
(CH2)��
(CH2)��
CH3
CH3
Figure 13.4.14 DiIC18(3)-DS (1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine-5,5’-disulfonic acid; D7776).
H3C
O3�
H3C
CH3
(CH
N
CH)2 CH
CH3
�O3H
N
(CH2)��
(CH2)��
CH3
CH3
Figure 13.4.15 DiIC18(5)-DS (1,1’-dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine-5,5’disulfonic acid; D12730).
CH3N
CH
CH
N�(CH2)�CH3�2
�
Figure 13.4.16 4-(4-(Didecylamino)styryl)-N-methylpyridinium iodide (4-Di-10-ASP, D291).
Figure 13.4.17 DiA; 4-Di-16-ASP (4-(4-(dihexadecylamino)styryl)-N-methylpyridinium
iodide; D3883).
CH3N
Dialkylaminostyryl Probes
H3C
CH3
CH
CH
N�(CH2)�CH CH CH2 CH CH(CH2)�CH3�2
O3�
C�
Figure 13.4.18 4-(4-(Dilinoleylamino) styryl)-N-methylpyridinium 4-chlorobenzenesulfonate (FAST DiA™ solid; DiΔ9,12-C18ASP, CBS; D7758).
™
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A Guide
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IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on
IMPORTANT NOTICE
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Chapter 13 — Probes for Lipids and Membranes
Section 13.4 Dialkylcarbocyanine and Dialkylaminostyryl Probes
Figure 13.4.19 Fluorescence excitation and emission spectra of DiA bound to phospholipid
bilayer membranes.
Figure 13.4.20 N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide (FM® 1-43, T3163).
tails and their fluorophore oriented parallel to the phospholipid acyl
chains.44 When these dialkylaminostyryl probes bind to membranes,
they exhibit a strong fluorescence enhancement; their fluorescence
in water is minimal. The interfacial solvation of the aminostyryl fluorophore causes a large blue shift of the absorption spectrum of the
membrane-bound probe.44 For example, the absorption maximum of
DiA is 456 nm when incorporated into DOPC liposomes and 490 nm
when in methanol. The fluorescence emission maximum of DiA in the
membrane environment is 590 nm, which is quite close to that observed
for probes with shorter alkyl tails such as 4-Di-10-ASP;44 however, the
fluorescence spectrum of DiA is very broad, with appreciable intensity
from about 510 nm to 690 nm (Figure 13.4.19). Consequently, DiA can
be detected as green, orange or even red fluorescence, depending on the
optical filter employed. Like the lipophilic carbocyanines, DiA is commonly used for neuronal membrane tracing (Section 14.4). FAST DiA™
(D7758), the diunsaturated analog of DiA, is intended to facilitate these
studies by accelerating dye diffusion within the membrane.
The FM® 1-43 (Figure 13.4.20), FM® 1-43FX, FM® 4-64 and FM® 5-95
dyes, which are discussed in detail in Section 16.1, are styryl dyes that
also exhibit high Stokes shifts and broad fluorescence emission but have
less lipophilic character than the 4-Di-10-ASP and DiA probes. The
FM® dyes are commonly used to define the outer membranes of liposomes and live cells and to detect synaptosome recycling.
REFERENCES
1. Trends Neurosci (1989) 12:333, 340; 2. Histochemistry (1992) 97:329; 3. Brain Res
(2008) 1215:11; 4. J Biomed Opt (2009) 14:054005; 5. Biochemistry (1974) 13:3315;
6. Chem Phys Lipids (2001) 109:175; 7. Biochemistry (1985) 24:5176; 8. J Cell Biol
(1985) 100:1309; 9. J Histochem Cytochem (1984) 32:608; 10. J Am Chem Soc (2007)
129:5798; 11. Bioorg Med Chem Lett (1996) 6:1479; 12. Biochim Biophys Acta (2000)
1467:227; 13. J Assoc Res Otolaryngol (2002) 3:289; 14. Biophys J (1979) 26:557;
15. J Cell Biol (1986) 103:807; 16. Biochemistry (1985) 24:582; 17. Nature (1981)
294:718; 18. Biochemistry (1980) 19:6199; 19. Biochim Biophys Acta (1994) 1191:164;
20. Biochim Biophys Acta (2000) 1467:101; 21. Biochim Biophys Acta (1990) 1023:25;
22. Biophys J (1994) 67:427; 23. Biophys J (1993) 65:568; 24. Biochemistry (1990)
29:8337; 25. J Membr Biol (1989) 109:221; 26. Biochim Biophys Acta (1983) 735:243;
27. J Cell Biol (1993) 121:543; 28. Chem Phys Lett (1989) 159:231; 29. FEBS Lett (2000)
467:52; 30. J Biol Chem (1994) 269:14614; 31. Biophys J (1993) 65:1019; 32. Analyst
(1990) 115:353; 33. Biochemistry (1977) 16:3836; 34. Biophys J (1998) 75:1131;
35. Biophys J (1995) 68:766; 36. Bioessays (1987) 6:117; 37. Chem Phys Lipids (1994)
73:139; 38. J Cell Biol (1991) 112:1143; 39. Biochim Biophys Acta (1992) 1107:193;
40. J Cell Physiol (1994) 158:7; 41. J Membr Biol (1993) 131:115; 42. J Cell Biol (1994)
125:795; 43. Science (1990) 247:1229; 44. Biophys J (1981) 34:353.
DATA TABLE 13.4 DIALKYLCARBOCYANINE AND DIALKYLAMINOSTYRYL PROBES
Cat. No.
MW
Storage
Soluble
Abs
EC
Em
Solvent
Notes
C7000
1051.50
F,D,L
DMSO, EtOH
553
134,000
570
MeOH
C7001
1051.50
F,D,L
DMSO, EtOH
553
134,000
570
MeOH
D275
881.72
L
DMSO, DMF
484
154,000
501
MeOH
D282
933.88
L
DMSO, EtOH
549
148,000
565
MeOH
D291
618.73
L
DMSO, EtOH
492
53,000
612
MeOH
1
D307
959.92
L
DMSO, EtOH
644
260,000
665
MeOH
2
D383
765.56
L
DMSO, EtOH
549
144,000
565
MeOH
3
D384
877.77
L
DMSO, EtOH
549
148,000
565
MeOH
D1125
825.61
L
DMSO, DMF
484
156,000
501
MeOH
D3883
787.05
L
DMSO, EtOH
491
52,000
613
MeOH
1
D3886
925.49
F,L,AA
DMSO, EtOH
549
144,000
564
MeOH
2
D3898
873.65
F,L,AA
DMSO, DMF
484
138,000
499
MeOH
D3899
925.82
F,L,AA
DMSO, EtOH
549
143,000
564
MeOH
2
D3911
933.88
L
DMSO, EtOH
549
148,000
565
MeOH
D7756
1017.97
F,L,AA
DMSO, EtOH
549
148,000
564
MeOH
D7757
1052.08
L
DMSO, EtOH
644
193,000
663
MeOH
D7758
899.80
F,L,AA
DMSO, EtOH
492
41,000
612
MeOH
1
D7776
993.54
L
DMSO, EtOH
555
144,000
570
MeOH
D7777
1145.73
L
DMSO, EtOH
556
164,000
573
MeOH
D7778
1115.55
L
DMSO, EtOH
497
175,000
513
MeOH
D12730
1019.58
L
DMSO, EtOH
650
247,000
670
MeOH
D12731
1013.41
L
DMSO, EtOH
748
270,000
780
MeOH
For definitions of the contents of this data table, see “Using The Molecular Probes® Handbook” in the introductory pages.
Notes
1. Abs and Em of styryl dyes are at shorter wavelengths in membrane environments than in reference solvents such as methanol. The difference is typically 20 nm for absorption and 80 nm for
emission, but varies considerably from one dye to another. Styryl dyes are generally nonfluorescent in water.
2. This product is intrinsically a liquid or an oil at room temperature.
3. This product is intrinsically a sticky gum at room temperature.
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Chapter 13 — Probes for Lipids and Membranes
Section 13.5 Other Nonpolar and Amphiphilic Probes
PRODUCT LIST 13.4 DIALKYLCARBOCYANINE AND DIALKYLAMINOSTYRYL PROBES
Cat. No.
Product
C7001
CellTracker™ CM-DiI
Quantity
C7000
CellTracker™ CM-DiI *special packaging*
D291
4-(4-(didecylamino)styryl)-N-methylpyridinium iodide (4-Di-10-ASP)
D383
1,1’-didodecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiIC12(3))
D3883
4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide (DiA; 4-Di-16-ASP)
D1125
3,3’-dihexadecyloxacarbocyanine perchlorate (DiOC16(3))
D384
1,1’-dihexadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiIC16(3))
D7758
4-(4-(dilinoleylamino)styryl)-N-methylpyridinium 4-chlorobenzenesulfonate (FAST DiA™ solid; DiΔ9,12-C18ASP, CBS)
D3898
3,3’-dilinoleyloxacarbocyanine perchlorate (FAST DiO™ solid; DiOΔ9,12-C18(3), ClO4)
5 mg
D7756
1,1’-dilinoleyl-3,3,3’,3’-tetramethylindocarbocyanine, 4-chlorobenzenesulfonate (FAST DiI™ solid; DiIΔ9,12-C18(3), CBS)
5 mg
D3899
1,1’-dilinoleyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (FAST DiI™ oil; DiIΔ9,12-C18(3), ClO4)
5 mg
D7778
3,3’-dioctadecyl-5,5’-di(4-sulfophenyl)oxacarbocyanine, sodium salt (SP-DiOC18(3))
5 mg
D7777
1,1’-dioctadecyl-6,6’-di(4-sulfophenyl)-3,3,3’,3’-tetramethylindocarbocyanine (SP-DiIC18(3))
D275
3,3’-dioctadecyloxacarbocyanine perchlorate (‘DiO’; DiOC18(3))
D7776
1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine-5,5’-disulfonic acid (DiIC18(3)-DS)
D282
1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (‘DiI’; DiIC18(3))
1 mg
20 x 50 µg
25 mg
100 mg
25 mg
25 mg
100 mg
5 mg
5 mg
100 mg
5 mg
100 mg
D3911
1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate *crystalline* (‘DiI’; DiIC18(3))
25 mg
D7757
1,1’-dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (‘DiD’ solid; DiIC18(5) solid)
10 mg
D12730
1,1’-dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine-5,5’-disulfonic acid (DiIC18(5)-DS)
5 mg
D307
1,1’-dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine perchlorate (‘DiD’ oil; DiIC18(5) oil)
25 mg
D12731
1,1’-dioctadecyl-3,3,3’,3’-tetramethylindotricarbocyanine iodide (‘DiR’; DiIC18(7))
10 mg
D3886
1,1’-dioleyl-3,3,3’,3’-tetramethylindocarbocyanine methanesulfonate (Δ9-DiI)
25 mg
L7781
Lipophilic Tracer Sampler Kit
1 kit
13.5 Other Nonpolar and Amphiphilic Probes
Amphiphilic Rhodamine, Fluorescein
and Coumarin Derivatives
Each of our amphiphilic probes comprises a moderately polar fluorescent dye with a lipophilic "tail." When used to stain membranes, including liposomes, the lipophilic portion of the
probe tends to insert in the membrane and the polar fluorophore resides on the surface, where
it senses the membrane’s surface environment and the surrounding medium.1 Our lipophilic
carbocyanines and styryl dyes (Section 13.4) are also amphiphilic molecules with a similar binding mode.
This section includes the classic membrane probes DPH, TMA-DPH, ANS, bis-ANS, TNS,
prodan, laurdan and nile red, and also some lipophilic BODIPY® and Dapoxyl® dyes developed in
our laboratories. Although they bear little resemblance to natural products, these probes tend to localize within cell membranes or liposomes or at their aqueous interfaces, where they are often used
to report on characteristics of their local environment, such as viscosity, polarity and lipid order.
Figure 13.5.1 Octadecyl rhodamine B chloride (O246).
Octadecyl Rhodamine B
The relief of the fluorescence self-quenching of octadecyl rhodamine B (O246, Figure 13.5.1)
can be used to monitor membrane fusion 2–7—one of several experimental approaches developed
for this application (Lipid-Mixing Assays of Membrane Fusion—Note 13.1). Octadecyl rhodamine B has been reported to undergo a potential-dependent "flip-flop" from one monolayer of
a fluid-state phospholipid bilayer membrane to the other, with partial relief of its fluorescence
quenching.8,9 Investigators have used octadecyl rhodamine B in conjunction with video microscopy 10–12 or digital imaging techniques 13 to monitor viral fusion processes. Membrane fusion can
™
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andand
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A Guide
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Probes
Labeling
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Chapter 13 — Probes for Lipids and Membranes
HO
O
O
C OH
O
NH C
O
(CH2)��CH3
Figure 13.5.2 5-Hexadecanoylaminofluorescein (H110).
Section 13.5 Other Nonpolar and Amphiphilic Probes
also be followed by monitoring fluorescence resonance energy transfer to octadecyl rhodamine
B from an acylaminofluorescein donor such as 5-hexadecanoylaminofluorescein 5,7,14,15 (H110,
Figure 13.5.2).
Fluorescence resonance energy transfer from fluorescein or dansyl labels to octadecyl rhodamine B has been used for structural studies of the blood coagulation factor IXa, EGF receptor
and receptor-bound IgE.16–18 Octadecyl rhodamine B has also been used to stain kinesin-generated membrane tubules,19 to characterize detergent micelles,20 to assay for lysosomal degradation of lipoproteins 21 and to investigate the influence of proteins on lipid dynamics using
time-resolved fluorescence anisotropy.22
Amphiphilic Fluoresceins
HO
O
O
C O(CH2)��CH3
O
Figure 13.5.3 Fluorescein octadecyl ester (F3857).
The amphiphilic fluorescein probes bind to membranes with the fluorophore at the aqueous
interface and the alkyl tail protruding into the lipid interior. 5-Dodecanoylaminofluorescein
(D109) is the hydrolysis product of our ImaGene Green™ C12-FDG β-galactosidase substrate
(D2893, Section 10.2). We also offer the homologous membrane probe 5-hexadecanoylaminofluorescein 1,15,23 (H110, Figure 13.5.2) and the octadecyl ester of fluorescein 24,25 (F3857,
Figure 13.5.3).
Amphiphilic fluorescein probes are commonly used for fluorescence recovery after photobleaching (FRAP) measurements of lipid lateral diffusion.26 Some researchers have reported that
5-hexadecanoylaminofluorescein stays predominantly in the outer membrane leaflet of epithelia
and does not pass through tight junctions, whereas the dodecanoyl derivative can "flip-flop"
to the inner leaflet at 20°C (but not at <10°C) and may also pass through tight junctions.27,28
More recent studies have indicated that the lack of tight junction penetration of 5-hexadecanoylaminofluorescein is due to probe aggregation rather than a significant difference in its
transport properties.29
Amphiphilic Coumarin
Figure 13.5.4 4-Heptadecyl-7-hydroxycoumarin (H22730).
Figure 13.5.5 DPH (1,6-diphenyl-1,3,5-hexatriene; D202).
4-Heptadecyl-7-hydroxycoumarin (H22730, Figure 13.5.4) is an alkyl derivative of the
pH-sensitive blue-fluorescent 7-hydroxycoumarin (umbelliferone) fluorophore. As with other
amphiphilic coumarins, 30 4-heptadecyl-7-hydroxycoumarin is primarily useful as a probe of
membrane surfaces. Deprotonation of the 7-hydroxyl group is expected to be strongly dependent
on membrane-surface electrostatic potential. The pKa of 4-heptadecyl-7-hydroxycoumarin varies from 6.35 in the cationic detergent CTAB to 11.15 in the anionic detergent sodium dodecyl
sulfate (SDS), as measured by its fluorescence response.31 However, its pKa in lipid assemblies is
strongly dependent on the ionic composition of the membrane surface, 31,32 making it a sensitive
probe of membrane-surface electrostatic potential.33 4-Heptadecyl-7-hydroxycoumarin has been
used to measure pH differences at membrane interfaces in isolated plasma membranes of normal
and multidrug-resistant murine leukemia cells.34,35 4-Heptadecyl-7-hydroxycoumarin has also
been employed as a structural probe for the head-group region of phospholipid bilayers. 36
DPH and DPH Derivatives
Diphenylhexatriene (DPH)
Figure 13.5.6 TMA-DPH (1-(4-trimethylammoniumphenyl)6-phenyl-1,3,5-hexatriene p-toluenesulfonate; T204).
Figure 13.5.7 4,4-Difluoro-1,3,5,7,8-pentamethyl-4-bora3a,4a-diaza-s-indacene (BODIPY® 493/503, D3922).
1,6-Diphenyl-1,3,5-hexatriene (DPH, D202; Figure 13.5.5) continues to be a popular fluorescent probe of membrane interiors. We also offer the cationic DPH derivative TMA-DPH, as well
as the phospholipid analog (D476, Section 13.2). The orientation of DPH within lipid bilayers is
loosely constrained. It is generally assumed to be oriented parallel to the lipid acyl chain axis,
but it can also reside in the center of the lipid bilayer parallel to the surface, as demonstrated
by time-resolved fluorescence anisotropy and polarized fluorescence measurements of oriented
samples.37–40 DPH shows no partition preference between coexisting gel- and fluid-phase phospholipids.41 Intercalation of DPH and its derivatives into membranes is accompanied by strong
enhancement of their fluorescence; their fluorescence is practically negligible in water. The fluorescence decay of DPH in lipid bilayers is complex.42–44 Fluorescence decay data are often analyzed in terms of continuous lifetime distributions,45–48 which are in turn interpreted as being
indicative of lipid environment heterogeneity.
DPH and its derivatives are cylindrically shaped molecules with absorption and fluorescence emission transition dipoles aligned approximately parallel to their long molecular
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Chapter 13 — Probes for Lipids and Membranes
Section 13.5 Other Nonpolar and Amphiphilic Probes
axis. Consequently, their fluorescence polarization is high in the absence of rotational motion and is very sensitive to reorientation of the
long axis resulting from interactions with surrounding lipids. These
properties have led to their extensive use for membrane fluidity measurements.49 The exact physical interpretation of these measurements
has some contentious aspects. For instance, the probes are largely sensitive to only the angular reorientation of lipid acyl chains—a motion
that does not necessarily correlate with other dynamic processes such
as lateral diffusion.50 Reviews on this subject 39,49,51,52 should be consulted for further discussion. Time-resolved fluorescence polarization
measurements of lipid order are more physically rigorous because they
allow the angular range of acyl chain reorientation ("lipid order") to be
resolved from its rate, and considerable research has been devoted to
the interpretation of these measurements.37,45,53,54
TMA-DPH
Designed to improve the localization of DPH in the membrane,
TMA-DPH (T204, Figure 13.5.6) contains a cationic trimethylammonium substituent that acts as a surface anchor.55–57 Like DPH, this derivative readily partitions from aqueous dispersions into membranes
and other lipid assemblies, accompanied by strong fluorescence enhancement. The lipid–water partition coefficient (Kp) for TMA-DPH
(Kp = 2.4 × 105) is lower than for DPH (Kp = 1.3 × 106), reflecting the
increased water solubility caused by the polar substituents.58 The fluorescence decay lifetime of TMA-DPH is more sensitive to changes in
lipid composition and temperature than is the fluorescence decay lifetime of DPH.59–61
Staining of cell membranes by TMA-DPH is much more rapid than
staining by DPH; however, the duration of plasma membrane surface
staining by TMA-DPH before internalization into the cytoplasm is
quite prolonged.62,63 As a consequence, TMA-DPH introduced into
Madin–Darby canine kidney (MDCK) cell plasma membranes does
not diffuse through tight junctions and remains in the apical domain,
whereas the anionic DPH propionic acid accumulates rapidly in intracellular membranes.64 TMA-DPH residing in the plasma membrane
can be extracted by washing with medium, thus providing a method
for isolating internalized probe and monitoring endocytosis 65 (Section
16.1). Furthermore, because TMA-DPH is virtually nonfluorescent in
water and binds in proportion to the available membrane surface,66
its fluorescence intensity is sensitive to increases in plasma membrane
surface area resulting from exocytosis.65,67,68
TMA-DPH fluorescence polarization measurements can be combined with video microscopy to provide spatially resolved images of
phospholipid order in large liposomes and single cells.69–72 Information
regarding lipid order heterogeneity among cell populations can be obtained in a similar way using flow cytometry.73–75
Nonpolar BODIPY® Probes
for oils and other nonpolar liquids. In addition, their photostability
is generally high; this, together with other favorable characteristics
(very low triplet–triplet absorption), make the BODIPY® 493/503 and
BODIPY® 505/515 fluorophores excellent choices for flashlamp-pumped
laser dyes.78,79
Staining with the BODIPY® 493/503 dye (D3922, Figure 13.5.7)
has been shown by flow cytometry to be more specific for cellular lipid droplets than staining with nile red 80 (N1142). The low molecular
weight of the BODIPY® 493/503 dye (262 daltons) results in the probe
having a relatively fast diffusion rate in membranes.81 The BODIPY®
493/503 dye has also been used to detect neutral compounds in a microchip channel separation device.82
BODIPY® 505/515 (D3921, Figure 13.5.8) rapidly permeates cell
membranes of live zebrafish embryos,83,84 selectively staining cytoplasmic yolk platelets. This staining provides dramatic contrast enhancement of cytoplasm relative to nucleoplasm and interstitial spaces, allowing individual cell boundaries and cell nuclei to be imaged clearly
with a confocal laser-scanning microscope (Figure 13.5.9).
The very long–wavelength BODIPY® 665/676 dye (B3932, Figure
13.5.10) has fluorescence that is not visible to the human eye; however,
it has found use as a probe for reactive oxygen species 85 (Section 18.2).
CH3
H3C
N
F
H3C
B
N
F
CH3
Figure 13.5.8 4,4-Difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY®
505/515, D3921).
Figure 13.5.9 Dorsal view of the midbrain/hindbrain region of a 15-somite stage zebrafish
embryo labeled with BODIPY® 505/515 (D3921). BODIPY® 505/515 localizes in lipidic yolk
platelets, producing selective cytoplasmic staining. This pseudocolored confocal image was
obtained using a Bio-Rad® MRC-600 microscope. Image contributed by Mark Cooper, University of Washington.
BODIPY® Fluorophores
BODIPY® fluorophore derivatives offer an unusual combination of
nonpolar structure (Figure 13.5.7) and long-wavelength absorption and
fluorescence.76 BODIPY® dyes have small fluorescence Stokes shifts, extinction coefficients that are typically greater than 80,000 cm–1M–1 and
high fluorescence quantum yields that are not diminished in water.77
These dyes have applications as stains for neutral lipids and as tracers
N
CH CH CH CH F
B
N
F
CH CH CH CH
Figure 13.5.10 (E,E)-3,5-bis-(4-phenyl-1,3-butadienyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY® 665/676, B3932).
™
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Chapter 13 — Probes for Lipids and Membranes
Section 13.5 Other Nonpolar and Amphiphilic Probes
BODIPY® FL C5-Ceramide
BODIPY® FL C5-ceramide (D3521, B22650; Section 13.3) stains the plasma membrane,
Golgi apparatus and cytoplasmic particles within the superficial enveloping layer (EVL) of embryos. Once the fluorescent lipid percolates through the EVL epithelium, however, it remains
localized within the interstitial fluid of the embryo and freely diffuses between cells (Figure
13.5.11). Vital staining with BODIPY® FL C5-ceramide thus allows hundreds of cells to be imaged
en masse during morphogenetic movements.83,86
CellTrace™ BODIPY® TR Methyl Ester
Figure 13.5.11 Cells in the notochord rudiment of a zebrafish
embryo undergoing mediolateral intercalation to lengthen the
forming notochord. BODIPY® FL C5-ceramide (D3521) localizes
in the interstitial fluid of the zebrafish embryo and freely diffuses
between cells, illuminating cell boundaries. This confocal image was
obtained using a Bio-Rad® MRC-600 microscope. Image contributed by Mark Cooper, University of Washington.
N
F
B
N
F
�
O
OCH2 C OCH3
Figure 13.5.12 CellTrace™ BODIPY® TR methyl ester (C34556).
CellTrace™ BODIPY®
TR methyl ester
Absorption
Fluorescence emission
EGFP
300
350
400
450
500
550
600
650
700
750
Wavelength (nm)
Figure 13.5.13 Normalized absorption (—) and fluorescence emission (– – –) spectra of enhanced Green Fluorescent Protein (EGFP) and CellTrace™ BODIPY® TR methyl ester
(C34556).
Many research and biotechnological applications require detailed three- and four-dimensional
visualization of embryonic cells labeled with Green Fluorescent Protein (GFP) within their native
tissue environments. Fluorescent counterstains that label all the cells in a living embryo provide a
histological context for the GFP-expressing cells in the specimen. The red-fluorescent CellTrace™
BODIPY® TR methyl ester (C34556, Figure 13.5.12) is an excellent counterstain for cells and tissues
that are expressing GFP.87 This dye readily permeates cell membranes and selectively stains mitochondria and endomembranous organelles such as endoplasmic reticulum and the Golgi apparatus,
but does not appear to localize in the plasma membrane. These localization properties make the dye
an ideal vital stain that can be used to reveal: (1) the location and shapes of cell nuclei, (2) the shapes
of cells within embryonic tissues and (3) the boundaries of organ-forming tissues within the whole
embryo.87 Furthermore, CellTrace™ BODIPY® TR methyl ester staining is retained after formaldehyde fixation and permeabilization with Triton X-100, and the dye does not appear to produce any
teratogenic effects on embryonic development. The emission spectra of enhanced GFP (EGFP) and
CellTrace™ BODIPY® TR methyl ester are well separated, with peaks at 508 nm and 625 nm, respectively (Figure 13.5.13), allowing simultaneous dual-channel confocal imaging without significant
overspill of GFP fluorescence into the CellTrace™ BODIPY® TR methyl ester detection channel.
The Image-iT® LIVE Intracellular Membrane and Nuclear Labeling Kit (I34407, Section 14.4)
provides the red-fluorescent CellTrace™ BODIPY® TR methyl ester along with the blue-fluorescent
Hoechst 33342 dye for highly selective staining of the intracellular membranes and nuclei, respectively, of live or fixed cells or tissues (Figure 13.5.14). These two fluorescent stains were especially
chosen for their compatibility with live GFP-expressing cells, and they can be combined into
one staining solution to save labeling time and wash steps while still providing optimal staining.
Pyrene, Nile Red and Bimane Probes
Nonpolar Pyrene Probe
1,3-Bis-(1-pyrene)propane (B311, Figure 13.5.15) has two pyrene moieties linked by a threecarbon alkylene spacer. This probe is somewhat analogous to the bis-pyrenyl phospholipids
(Section 13.2) in that excimer formation (and, consequently, the fluorescence emission wavelength) is controlled by intramolecular rather than bimolecular interactions. Thus, this probe is
highly sensitive to constraints imposed by its environment, and can therefore be used as a viscosity sensor for interior regions of lipoproteins, membranes, micelles, liquid crystals and synthetic
polymers.88 Because excimer formation results in a spectral shift (Figure 13.5.16), the probe may
be useful for ratio imaging of molecular mobility.89 However, pyrene fatty acids (Section 13.2)
appear to be preferable for this purpose because the uptake of 1,3-bis-(1-pyrene)propane by cells
is limited.
Nile Red
Figure 13.5.14 Live HeLa cells were transfected using
pShooter™ vector pCMV/myc/mito/GFP and Lipofectamine®
2000 transfection reagent and stained with the reagents
in the Image-iT® LIVE Intracellular Membrane and Nuclear
Labeling Kit (I34407). Intracellular membranes were stained
with CellTrace™ BODIPY® TR methyl ester, and nuclei were
stained with Hoechst 33342. Cells were visualized using
epifluorescence microscopy.
The phenoxazine dye nile red (N1142, Figure 13.5.17) is used to localize and quantitate lipids, particularly neutral lipid droplets within cells.80,90–92 It is selective for neutral lipids such as
cholesteryl esters 93,94 (and also, therefore, for lipoproteins) and is suitable for staining lysosomal
phospholipid inclusions.95 Nile red is almost nonfluorescent in water and other polar solvents but
undergoes fluorescence enhancement and large absorption and emission blue shifts in nonpolar
environments.96,97 Its fluorescence enhancement upon binding to proteins is weaker than that
produced by its association with lipids 97 (Figure 13.5.18). Ligand-binding studies on tubulin
and tryptophan synthase 98 have exploited the environmental sensitivity of nile red’s fluorescence. Nile red has also been used to detect sphingolipids on thin-layer chromatograms 99 and to
stain proteins after SDS-polyacrylamide gel electrophoresis.100
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Chapter 13 — Probes for Lipids and Membranes
Section 13.5 Other Nonpolar and Amphiphilic Probes
Bimane Azide
(CH2)3
Bimane azide (B30600, Figure 13.5.19) is a small blue-fluorescent photoreactive alkyl azide
(excitation/emission maxima ~375/458 nm) for photoaffinity labeling of proteins, potentially
including membrane proteins from within the cell membrane. This reactive fluorophore’s small
size may reduce the likelihood that the label will interfere with the function of the biomolecule,
an important advantage for site-selective probes.
Figure 13.5.15 1,3-Bis-(1-pyrenyl)propane (B311).
Steatosis, the intracellular accumulation of neutral lipids as lipid droplets or globules, is often
triggered by drugs that affect the metabolism of fatty acids or neutral lipids. LipidTOX™ neutral
lipid stains were developed to characterize the effects of drugs and other compounds on lipid
metabolism in mammalian cell lines. LipidTOX™ neutral lipid stains have an extremely high
affinity for neutral lipid droplets. These reagents are added after cell fixation and do not require
subsequent wash steps after incubation with the sample. Key advantages of this series of neutral
lipid stains over conventional stains such as nile red include their ready-to-use formulations,
their flexibility for multiplexing protocols and their compatibility with LipidTOX™ phospholipid
stains (H34350, H34351; Section 13.2).
LipidTOX™ neutral lipid stains are available with green, red and deep red fluorescence
emission:
• HCS LipidTOX™ Green neutral lipid stain (H34475), with excitation/emission maxima
~495/505 nm (Figure 13.5.20)
• HCS LipidTOX™ Red neutral lipid stain (H34476), with excitation/emission maxima
~577/609 nm
• HCS LipidTOX™ Deep Red neutral lipid stain (H34477), with excitation/emission maxima
~637/655 nm
A
Ex = 370 nm
B
DOPC
1
2
3
4
350
400
450
450
500
550
Wavelength (nm)
600
550
600
Figure 13.5.16 Excimer formation by pyrene in
ethanol. Spectra are normalized to the 371.5 nm peak of the
monomer. All spectra are essentially identical below 400 nm
after normalization. Spectra are as follows: 1) 2 mM pyrene,
purged with argon to remove oxygen; 2) 2 mM pyrene, airequilibrated; 3) 0.5 mM pyrene (argon-purged); and 4) 2 µM
pyrene (argon-purged). The monomer-to-excimer ratio
(371.5 nm/470 nm) is dependent on both pyrene concentration and the excited-state lifetime, which is variable because
of quenching by oxygen.
Ex = 540 nm
BSA
Figure 13.5.17 Nile red (N1142).
O
400
500
Wavelength (nm)
DOPC
Fluorescence emission
Fluorescence emission
BSA
Fluorescence emission
LipidTOX™ Neutral Lipid Stains
550
600
650
700
750
CH3
Wavelength (nm)
Figure 13.5.18 Fluorescence emission spectra of A) 1,8-ANS (A47) and B) nile red (N1142) bound to protein and phospholipid
vesicles. Samples comprised 1 µM dye added to 20 µM bovine serum albumin (BSA) or 100 µM dioctadecenoylglycerophosphocholine (DOPC).
CH3
O
N
N
CH3
CH2N N N
Figure 13.5.19 Bimane azide (B30600).
Figure 13.5.20 FABP4 antibody labeling in adipocytes. Adipocytes differentiated from 3T3-L1 mouse fibroblasts were fixed with formaldehyde and permeabilized
with saponin before labeling with rabbit anti–fatty acid
binding protein (FABP4) IgG (red). These cells were then
stained with LipidTOX™ Green neutral lipid stain (H34475,
green), counterstained with DAPI (D1306, D21490; blue) and
mounted in ProLong® Gold antifade reagent (P36930).
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Chapter 13 — Probes for Lipids and Membranes
Figure 13.5.21 Prodan (6-propionyl-2-dimethylaminonaphthalene; P248).
Figure 13.5.22 Normalized emission spectra of prodan
(P248) excited at 345 nm in 1) cyclohexane, 2) dimethylformamide, 3) ethanol and 4) water.
Figure 13.5.23 6-Dodecanoyl-2-dimethylaminonaphthalene (laurdan, D250).
Figure 13.5.24 Absorption and fluorescence emission
spectra of Dapoxyl® (2-aminoethyl)sulfonamide in methanol.
Figure 13.5.25 Dapoxyl® sulfonic acid, sodium salt (D12800).
Section 13.5 Other Nonpolar and Amphiphilic Probes
These HCS LipidTOX™ neutral lipid stains have been used to image intracellular lipid accumulation in rat cortical neurons, COS-7 cells and hepatitis C virus (HCV)–infected FT3-7 human
hepatoma cells.101–103 HCS LipidTOX™ Red neutral lipid stain was used to detect RNAi knockdown
of acyl-coenzyme A:cholesterol acyl transferase, isoform 1 (ACAT-1), an endoplasmic reticulum
enzyme that regulates the equilibrium between free cholesterol and cholesteryl esters in cells.104
LipidTOX™ Green neutral lipid stain is also a component of the HCS LipidTOX™
Phospholipidosis and Steatosis Detection Kit (H34157, H34158; Section 13.2), which provides a
complete set of reagents for performing high-content screening (HCS) assays to detect and distinguish the intracellular accumulation of phospholipids (phospholipidosis) and of neutral lipids
(steatosis) in mammalian cell lines after exposure to test compounds. In addition, HCS LipidTOX™
neutral lipid stains can be used to monitor the formation and differentiation of adipocytes, a process called adipogenesis. Adipogenesis is of acute interest to the biomedical and drug discovery
community as it plays an important role in diseases such as obesity, diabetes and atherosclerosis.
HCS LipidTOX™ neutral lipid stains are designed for fixed–end point workflows in which
formaldehyde-fixed cells in microplates are processed, imaged and analyzed. These stains can easily be detected with fluorescence microscopes or HCS readers equipped with standard filter sets.
Membrane Probes with Environment-Sensitive Spectral
Shifts
Prodan and Laurdan
Prodan (P248, Figure 13.5.21), introduced by Weber and Farris in 1979, has both electrondonor and electron-acceptor substituent, resulting in a large excited-state dipole moment and
extensive solvent polarity–dependent fluorescence shifts 105 (Figure 13.5.22). Several variants of the
original probe have since been prepared, including the lipophilic derivative laurdan (D250, Figure
13.5.23) and thiol-reactive derivatives acrylodan and badan (A433, B6057; Section 2.3), which
can be used to confer the environment-sensitive properties of this fluorophore on bioconjugates.
When prodan or its derivatives are incorporated into membranes, their fluorescence spectra
are sensitive to the physical state of the surrounding phospholipids.106 In membranes, prodan
appears to localize at the surface,107 although Fourier transform infrared (FTIR) measurements
indicate some degree of penetration into the lipid interior.108 Excited-state relaxation of prodan is
sensitive to the nature of the linkage (ester or ether) between phospholipid hydrocarbon tails and
the glycerol backbone.109 In contrast, laurdan’s excited-state relaxation is independent of headgroup type, and is instead determined by water penetration into the lipid bilayer.110,111 Two-photon
infrared excitation techniques have been successfully applied to both prodan and laurdan, although both probes nominally require ultraviolet excitation 112–115 (~360 nm).
Much experimental work using these probes has sought to characterize coexisting lipid domains based on their distinctive fluorescence spectra,113,116–120 an approach that is intrinsically
amenable to dual-wavelength ratio measurements.111,121 Other applications include detecting nonbilayer lipid phases,122,123 mapping changes in membrane structure induced by cholesterol and
alcohols 124–127 and assessing the polarity of lipid/water interfaces.128,129 Like ANS, prodan is also
useful as a noncovalently interacting probe for proteins.130–133
Dapoxyl® Derivative
We have developed a variety of probes based on our Dapoxyl® fluorophore.134 Dapoxyl® sulfonamide derivatives exhibit UV absorption with maxima near 370 nm, extinction coefficients
>24,000 cm–1M–1 and Stokes shifts in excess of 200 nm (Figure 13.5.24). Dapoxyl® sulfonic acid
(D12800, Figure 13.5.25) is an amphiphilic Dapoxyl® derivative with generally similar properties
and applications to anilinonaphthalene sulfonate (ANS) (Monitoring Protein-Folding Processes
with Environment-Sensitive Dyes—Note 9.1). Both ANS and Dapoxyl® sulfonic acid have been
used in a drug-discovery assay based on the detection of protein thermal denaturation shifts.135
Reactive versions of the Dapoxyl® fluorophore are described in Section 1.7 and Section 3.4.
Anilinonaphthalenesulfonate (ANS) and Related Derivatives
The use of anilinonaphthalene sulfonates (ANS) as fluorescent probes dates back to the pioneering work of Weber in the 1950s, and this class of probes remains valuable for studying both membrane
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Chapter 13 — Probes for Lipids and Membranes
Section 13.5 Other Nonpolar and Amphiphilic Probes
surfaces and proteins. Slavik’s 1982 review of its properties is recommended reading,
especially for the extensive compilation of spectral data.136 The primary member of this
class, 1,8-ANS (A47, Figure 13.5.26), and its analogs 2,6-ANS (A50) and 2,6-TNS (T53)
are all essentially nonfluorescent in water, only becoming appreciably fluorescent when
bound to membranes (quantum yields ~0.25) or proteins (quantum yields ~0.7)136–138
(Figure 13.5.18). This property makes them sensitive indicators of protein folding, conformational changes 139–142 and other processes that modify the exposure of the probe to
water (Monitoring Protein-Folding Processes with Environment-Sensitive Dyes—Note
9.1). Fluorescence of 2,6-ANS is also enhanced by cyclodextrins, permitting a sensitive
method for separating and analyzing cyclodextrins with capillary electrophoresis.143
Figure 13.5.26 1,8-ANS (1-anilinonaphthalene-8-sulfonic
acid, A47).
Bis-ANS
Bis-ANS (B153, Figure 13.5.27) is superior to 1,8-ANS as a probe for nonpolar
cavities in proteins, often binding with an affinity that is orders-of-magnitude higher.144–147 Bis-ANS has particularly high affinity for nucleotide-binding sites of some
proteins.148–150 It is also useful as a structural probe for tubulin 151,152 and as an inhibitor of microtubule assembly.153–155 Covalent photoincorporation of bis-ANS into
proteins has been reported.156
Figure 13.5.27 bis-ANS (4,4’-dianilino-1,1’-binaphthyl-5,5’disulfonic acid, dipotassium salt; B153).
DCVJ
The styrene derivative DCVJ (D3923, Figure 13.5.28) is a sensitive indicator of
tubulin assembly and actin polymerization.157,158 The fluorescence quantum yield of
DCVJ is strongly dependent on environmental rigidity, resulting in large fluorescence
increases when the dye binds to antibodies 159 and when it is compressed in synthetic
polymers or phospholipid membrane interiors.160,161 DCVJ has been used for microviscosity measurements of phospholipid bilayers.161
CH C(CN)2
N
Figure 13.5.28 DCVJ (4-(dicyanovinyl)julolidine, D3923).
REFERENCES
1. Biochim Biophys Acta (1998) 1374:63; 2. Biophys J (1999) 77:943; 3. Photochem
Photobiol (1994) 60:563; 4. Biophys Chem (1989) 34:283; 5. Chem Phys Lipids (2002)
116:39; 6. Biochemistry (1984) 23:5675; 7. J Cell Sci (1977) 28:167; 8. Biophys J (1996)
71:2680; 9. Biochim Biophys Acta (1995) 1237:121; 10. Biophys J (1992) 63:710; 11. J Gen
Physiol (1991) 97:1101; 12. Proc Natl Acad Sci U S A (1990) 87:1850; 13. FEBS Lett
(1989) 250:487; 14. Biophys J (1999) 76:1812; 15. Biochim Biophys Acta (1994) 1189:175;
16. J Biol Chem (1992) 267:17012; 17. Biochemistry (1991) 30:9125; 18. Biochemistry
(1990) 29:8741; 19. J Cell Biol (1988) 107:2233; 20. Langmuir (1994) 10:658; 21. Eur
J Cell Biol (1992) 59:116; 22. Eur Biophys J (1990) 18:277; 23. FEBS Lett (1989) 257:10;
24. Cytometry (1996) 24:368; 25. Biophys J (1996) 70:988; 26. J Cell Sci (1991) 100:473;
27. Exp Cell Res (1989) 181:375; 28. Nature (1981) 294:718; 29. Biochem Biophys Res
Commun (1992) 184:160; 30. Biochim Biophys Acta (1996) 1284:191; 31. J Phys Chem
(1977) 81:1755; 32. Biochim Biophys Acta (1973) 323:326; 33. Methods Enzymol (1989)
171:376; 34. Biochemistry (1990) 29:7275; 35. Biochim Biophys Acta (1983) 729:185;
36. Biochemistry (1985) 24:573; 37. Biochemistry (1991) 30:5565; 38. Chem Phys Lipids
(1991) 57:39; 39. Biochimie (1989) 71:23; 40. Biochim Biophys Acta (1986) 859:209;
41. Biochim Biophys Acta (1988) 941:102; 42. Biophys Chem (1993) 48:205; 43. Biophys
J (1991) 59:466; 44. Biophys J (1989) 56:723; 45. Biophys Chem (1994) 48:337; 46. Biochim
Biophys Acta (1992) 1104:273; 47. Biochemistry (1990) 29:3248; 48. Chem Phys Lipids
(1989) 50:1; 49. Chem Phys Lipids (1993) 64:99; 50. Biochim Biophys Acta (1981) 649:471;
51. Chem Phys Lipids (1993) 64:117; 52. Biochim Biophys Acta (1986) 854:38; 53. Chem
Phys (1994) 185:393; 54. Chem Phys Lett (1993) 216:559; 55. Biochemistry (1998) 37:8180;
56. Biochemistry (1988) 27:7723; 57. Biochemistry (1981) 20:7333; 58. Biochem Biophys
Res Commun (1991) 181:166; 59. Chem Phys Lipids (1990) 55:29; 60. Biochemistry
(1987) 26:5121; 61. Biochemistry (1987) 26:5113; 62. Biochim Biophys Acta (1985)
845:60; 63. Cell Biophys (1983) 5:129; 64. Am J Physiol (1988) 255:F22; 65. Biochim
Biophys Acta (1995); 66. Biochemistry (1986) 25:2149; 67. J Cell Biol (1996) 135:1741;
68. Biochim Biophys Acta (1993) 1147:194; 69. Fluorescent and Luminescent Probes
for Biological Activity, Mason WT, Ed. (1993) p. 420; 70. Am J Physiol (1991) 260:C1;
71. FASEB J (1991) 5:2078; 72. Biophys J (1990) 57:1199; 73. Cytometry (2000) 39:151;
74. Biochim Biophys Acta (1991) 1067:71; 75. Plant Physiol (1990) 94:729; 76. J Photochem
Photobiol A (1999) 121:177; 77. Anal Biochem (1991) 198:228; 78. Heteroatomic Chem
(1990) 1:389; 79. Optics Comm (1989) 70:425; 80. Cytometry (1994) 17:151; 81. Biophys
J (1996) 71:2656; 82. Electrophoresis (2003) 24:3253; 83. Methods Mol Biol (1999) 122:185;
84. Neuron (1998) 20:1081; 85. J Agric Food Chem (2000) 48:1150; 86. Methods Cell Biol
(1999) 59:179; 87. Dev Dyn (2007) 232:359; 88. Biochim Biophys Acta (1993) 1149:86;
89. Eur J Cell Biol (1994) 65:172; 90. J Histochem Cytochem (1997) 45:743; 91. J Cell
Biol (1993) 123:1567; 92. Exp Cell Res (1992) 199:29; 93. J Cell Biol (1989) 108:2201;
94. J Chromatogr (1987) 421:136; 95. Histochemistry (1992) 97:349; 96. Anal Chem
(1990) 62:615; 97. Anal Biochem (1987) 167:228; 98. J Biol Chem (1995) 270:6357; 99. Anal
Biochem (1993) 208:121; 100. Biotechniques (1996) 21:625; 101. J Biol Chem (2009)
284:3049; 102. J Virol (2008) 82:7624; 103. J Biol Chem (2009) 284:2383; 104. FEBS Lett
(2007) 581:1688; 105. Photochem Photobiol (1993) 58:499; 106. Photochem Photobiol
(1999) 70:557; 107. Biochemistry (1988) 27:399; 108. Biochemistry (1989) 28:8358;
109. Biochemistry (1990) 29:11134; 110. Biophys J (1994) 66:763; 111. Biophys J (1991)
60:179; 112. Anal Chem (2001) 73:2302; 113. Biophys J (2000) 78:290; 114. Biophys J (1999)
77:2090; 115. Biophys J (1997) 72:2413; 116. Biophys J (2001) 80:1417; 117. Biophys J (1994)
66:120; 118. Photochem Photobiol (1993) 57:420; 119. Biophys J (1990) 57:1179; 120. J Biol
Chem (1990) 265:20044; 121. Photochem Photobiol (1993) 57:403; 122. Biochemistry
(1992) 31:1550; 123. Biophys J (1990) 57:925; 124. Biochim Biophys Acta (2001) 1511:330;
125. Biophys J (1995) 68:1895; 126. Biophys J (1993) 65:1404; 127. Biochemistry
(1992) 31:9473; 128. J Biol Chem (1994) 269:10298; 129. J Biol Chem (1994) 269:7429;
130. Biochemistry (1998) 37:7167; 131. Biochem J (1993) 290:411; 132. Eur J Biochem
(1992) 204:127; 133. Nature (1986) 319:70; 134. J Photochem Photobiol A (2000) 131:95;
135. J Biomol Screen (2001) 6:429; 136. Biochim Biophys Acta (1982) 694:1; 137. Biophys
J (1998) 74:422; 138. Biochemistry (1968) 7:3381; 139. Biochemistry (1999) 38:2110;
140. Biochemistry (1998) 37:4621; 141. Biochemistry (1998) 37:13862; 142. Biochemistry
(1998) 37:16802; 143. J Chromatogr A (1994) 680:233; 144. Arch Biochem Biophys
(1989) 268:239; 145. Biochemistry (1985) 24:3852; 146. Biochemistry (1985) 24:2034;
147. Biochemistry (1969) 8:3915; 148. Biochemistry (1992) 31:2982; 149. Biochim Biophys
Acta (1990) 1040:66; 150. Proc Natl Acad Sci U S A (1977) 74:2334; 151. Biochemistry
(1998) 37:4687; 152. Biochemistry (1994) 33:11891; 153. Biochemistry (1998) 37:17571;
154. Biochemistry (1992) 31:6470; 155. J Biol Chem (1984) 259:14647; 156. Biochemistry
(1995) 34:7443; 157. Anal Biochem (1992) 204:110; 158. Biochemistry (1989) 28:6678;
159. Biochemistry (1993) 32:7589; 160. Chem Phys (1993) 169:351; 161. Biochemistry
(1986) 25:6114.
™
The
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A Guide
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and
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IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on
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585
Chapter 13 — Probes for Lipids and Membranes
Section 13.5 Other Nonpolar and Amphiphilic Probes
DATA TABLE 13.5 OTHER NONPOLAR AND AMPHIPHILIC PROBES
Cat. No.
MW
Storage
Soluble
Abs
EC
Em
Solvent
Notes
A47
299.34
L
pH >6, DMF
372
7800
480
MeOH
1
A50
299.34
L
DMF
319
27,000
422
MeOH
1
B153
672.85
L
pH >6
395
23,000
500
MeOH
1, 2
344
80,000
378
MeOH
3
B311
444.57
L
MeCN, CHCl3
665
161,000
676
MeOH
4
B3932
448.32
F,L
DMSO, CHCl3
B30600
233.23
F,D,L
DMSO
375
6000
458
MeOH
C34556
438.25
F,D,L
DMSO
588
68,000
616
MeOH
5
D109
529.63
L
DMSO, EtOH
495
85,000
518
MeOH
6
D202
232.32
L
DMF, MeCN
350
88,000
452
MeOH
7, 8
D250
353.55
L
DMF, MeCN
364
20,000
497
MeOH
9
D3921
248.08
F,L
EtOH, DMSO
502
98,000
510
MeOH
4
D3922
262.11
F,L
EtOH, DMSO
493
89,000
504
MeOH
4
D3923
249.31
L
DMF, DMSO
456
61,000
493
MeOH
358
25,000
517
MeOH
10
D12800
366.37
L
DMSO, H2O
F3857
584.79
L
DMSO, EtOH
504
95,000
525
MeOH
6
H110
585.74
L
DMSO, EtOH
497
92,000
519
MeOH
6
H22730
400.60
L
DMSO, EtOH
366
20,000
453
MeOH
6
H34475
~300
F,L
DMSO
495
94,000
505
MeOH
5, 13
H34476
~400
F,L
DMSO
574
62,000
609
MeOH
5, 13
H34477
~350
F,L
DMSO
626
68,000
648
MeOH
5, 13
N1142
318.37
L
DMF, DMSO
552
45,000
636
MeOH
11
O246
731.50
F,DD,L
DMSO, EtOH
556
125,000
578
MeOH
12
P248
227.31
L
DMF, MeCN
363
19,000
497
MeOH
9
T53
335.35
L
DMF
318
26,000
443
MeOH
1
T204
461.62
D,L
DMF, DMSO
355
75,000
430
MeOH
7
For definitions of the contents of this data table, see “Using The Molecular Probes® Handbook” in the introductory pages.
Notes
1. Fluorescence quantum yields of ANS and its derivatives are environment dependent and are particularly sensitive to the presence of water. QY of A47 is about 0.4 in EtOH, 0.2 in MeOH and 0.004
in water. Em is also somewhat solvent dependent. (Biochim Biophys Acta (1982) 694:1)
2. B153 is soluble in water at 0.1–1.0 mM after heating.
3. Absorption spectra of bis-pyrenyl alkanes have additional peaks at ~325 nm and <300 nm. Emission spectra include both monomer (~380 nm and ~400 nm) and excimer (~470 nm) peaks.
4. The absorption and fluorescence spectra of BODIPY® derivatives are relatively insensitive to the solvent.
5. This product is supplied as a ready-made solution in the solvent indicated under "Soluble."
6. Spectra of this product are pH dependent. Data listed are for basic solutions prepared in methanol containing a trace of KOH.
7. Diphenylhexatriene (DPH) and its derivatives are essentially nonfluorescent in water. Absorption and emission spectra have multiple peaks. The wavelength, resolution and relative intensity of
these peaks are environment dependent. Abs and Em values are for the most intense peak in the solvent specified.
8. Stock solutions of DPH (D202) are often prepared in in tetrahydrofuran (THF). Long-term storage of THF solutions is not recommended because of possible peroxide formation in that solvent.
9. The emission spectrum of P248 is solvent dependent. Em = 401 nm in cyclohexane, 440 nm in CHCl3, 462 nm in MeCN, 496 nm in EtOH and 531 nm in H2O. (Biochemistry (1979) 18:3075) Abs is
only slightly solvent dependent. The emission spectra of D250 in these solvents are similar to those of P248.
10. Em = 520 nm when bound to phospholipid bilayer membranes. Fluorescence in H2O is weak (Em ~600 nm).
11. The absorption and fluorescence spectra and fluorescence quantum yield of N1142 are highly solvent dependent. (J Lipid Res (1985) 26:781, Anal Biochem (1987) 167:228)
12. This product is intrinsically a sticky gum at room temperature.
13. Abs/Em in trioctanylglycerol = 498/507 nm, 582/616 nm and 635/652 nm for H34475, H34476 and H34477 respectively.
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A Guide
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Fluorescent Probes
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586
IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on
IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
the Appendix on
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Chapter 13 — Probes for Lipids and Membranes
Section 13.5 Other Nonpolar and Amphiphilic Probes
PRODUCT LIST 13.5 OTHER NONPOLAR AND AMPHIPHILIC PROBES
Cat. No.
Product
A47
1,8-ANS (1-anilinonaphthalene-8-sulfonic acid) *high purity*
Quantity
100 mg
A50
2,6-ANS (2-anilinonaphthalene-6-sulfonic acid)
100 mg
B30600
Bimane azide
B153
bis-ANS (4,4’-dianilino-1,1’-binaphthyl-5,5’-disulfonic acid, dipotassium salt)
5 mg
B3932
(E,E)-3,5-bis-(4-phenyl-1,3-butadienyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY® 665/676)
B311
1,3-bis-(1-pyrenyl)propane
10 mg
5 mg
25 mg
C34556
CellTrace™ BODIPY® TR methyl ester *lipophilic counterstain for GFP* *solution in DMSO*
D12800
Dapoxyl® sulfonic acid, sodium salt
1 mL
10 mg
D3923
DCVJ (4-(dicyanovinyl)julolidine)
25 mg
D3922
4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY® 493/503)
10 mg
D3921
4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY® 505/515)
D109
5-dodecanoylaminofluorescein
100 mg
10 mg
D250
6-dodecanoyl-2-dimethylaminonaphthalene (laurdan)
100 mg
D202
DPH (1,6-diphenyl-1,3,5-hexatriene)
100 mg
F3857
Fluorescein octadecyl ester
10 mg
H34477
HCS LipidTOX™ Deep Red neutral lipid stain *solution in DMSO* *for cellular imaging*
125 µL
H34475
HCS LipidTOX™ Green neutral lipid stain *solution in DMSO* *for cellular imaging*
125 µL
H34476
HCS LipidTOX™ Red neutral lipid stain *solution in DMSO* *for cellular imaging*
125 µL
H22730
4-heptadecyl-7-hydroxycoumarin
10 mg
H110
5-hexadecanoylaminofluorescein
100 mg
N1142
Nile red
25 mg
O246
Octadecyl rhodamine B chloride (R18)
10 mg
P248
Prodan (6-propionyl-2-dimethylaminonaphthalene)
T204
TMA-DPH (1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate)
T53
2,6-TNS (2-(p-toluidinyl)naphthalene-6-sulfonic acid, sodium salt)
100 mg
25 mg
100 mg
™
The
Probes
Handbook:
A Guide
to Fluorescent
Probes
and
Labeling
Technologies
TheMolecular
Molecular
Probes®
Handbook:
A Guide
to Fluorescent
Probes
and
Labeling
Technologies
IMPORTANT
NOTICE:described
The products
described
thiscovered
manual are
by oneLimited
or moreUse
Limited
Use
Label License(s).
to the
Appendix
IMPORTANT NOTICE
: The products
in this
manualinare
by covered
one or more
Label
License(s).
PleasePlease
referrefer
to the
Appendix
on on
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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587