Download Probes for Multiplexed Detection of GFP

A Diverse Selection of Organelle Probes—Section 12.1
Molecular Probes offers a diverse array of cell-permeant fluorescent stains that selectively associate with the mitochondria,
lysosomes, endoplasmic reticulum and Golgi apparatus in live cells. These probes, which include our exclusive MitoTracker,
MitoFluor, LysoTracker, LysoSensor, RedoxSensor and ER-Tracker organelle stains (Molecular Probes' organelle-selective
probes—Table 12.1, Figure 12.1), are compatible with most fluorescence instrumentation and provide researchers with powerful
tools for investigating respiration, mitosis, apoptosis, multidrug resistance, substrate degradation and detoxification, intracellular
transport and sorting and more. Moreover, unlike antibodies, these fluorescent probes can be used to investigate organelle structure
and activity in live cells with minimal disruption of cellular function ( ). The red-fluorescent organelle stains are particularly
useful for demonstrating colocalization with green-fluorescent protein (GFP) expression
(Fluorescent Probes for Use with
GFP—Note 12.1). An excellent compendium of human diseases that affect intracellular transport processes through lysosomes,
Golgi apparatus and endoplasmic reticulum has been published.
A particularly useful general review of cell organelles and other
topics appears at the Cell Biology Topics web site (http://cellbio.utmb.edu/cellbio/).
Figure 12.1 Diagram of an animal cell.
We have also introduced a large collection of organelle-specific monoclonal antibodies for both mammalian and yeast cells that can
be used for immunolocalization, immunoprecipitation and Western blot analysis. Our antibodies to mitochondrial proteins
(Monoclonal antibodies specific for proteins in the oxidative phosphorylation system—Table 12.4, Monoclonal antibodies specific
for mitochondrial proteins not associated with the oxidative phosphorylation system—Table 12.6, Figure 12.33) provide a unique
set of tools for understanding the assembly and function of mitochondria. Cell-permeant and -impermeant fluorescent stains for the
nucleus are described in Nucleic Acid Detection and Genomics Technology—Chapter 8, probes for the cytoskeleton in Probes for
Cytoskeletal Proteins—Chapter 11, and plasma membrane stains in Probes for Lipids and Membranes—Chapter 13. A variety of
probes for phagovacuoles, endosomes and lysosomes—including membrane markers as well as ligands for studying receptormediated endocytosis—are discussed in Probes for Following Receptor Binding, Endocytosis and Exocytosis—Section 16.1.
Figure 12.33. Major protein complexes of the oxidative phosphorylation (OxPhos) system and antibodies that recognize them.
Molecular Probes' organelle-selective probes - Table 12.1
Probes for Mitochondria — Probes for Mitochondria - Section 12.2
Chemiluminescent Probe
L6868
Lucigenin †
Green-Fluorescent Probes
A1372
Nonyl acridine orange
D273
DiOC6(3)
D378
DiOC7(3) (for plant mitochondria)
M7502
MitoFluor Green
M7514
MitoTracker Green FM *
R302, R22420
Rhodamine 123
S7529
SYTO 18 yeast mitochondrial stain
T3168
JC-1 ‡
D22421
JC-9 ‡
Yellow- and Orange-Fluorescent Probes
D288
4-Di-1-ASP (DASPMI)
D426
DASPEI
M7510
MitoTracker Orange CMTMRos *
R634
Rhodamine 6G
R648MP
Rhodamine B, hexyl ester
T639
Tetramethylrosamine
T668
Tetramethylrhodamine, methyl ester
T669
Tetramethylrhodamine, ethyl ester
Red-Fluorescent Probes
M7512
MitoTracker Red CMXRos *
T3168
JC-1 ‡
D22421
JC-9 ‡
M22425
MitoTracker Red 580 *
M22424
MitoFluor Red 589
M22422
MitoFluor Red 594
M22426
MitoTracker Deep Red 633 *
* Aldehyde-fixable probe. † Chemiluminescent probe. ‡ Dual-emission spectrum.
Probes for Mitochondria (Probes Requiring Intracellular Oxidation) — Probes for Mitochondria - Section 12.2
Green-Fluorescent Probe
D632
Dihydrorhodamine 123
Yellow- and Orange-Fluorescent Probes
D633
Dihydrorhodamine 6G
M7511
MitoTracker Orange CM-H2TMRos *
Red-Fluorescent Probes
M7513
MitoTracker Red CM-H2XRos *
R14060
RedoxSensor Red CC-1 †
* Aldehyde-fixable probe. † The differential distribution of the oxidized product between mitochondria and lysosomes appears to
depend on the oxidation–reduction (redox) potential of the cytosol.
Probes for Acidic Organelles, Including Lysosomes — Probes for Lysosomes, Peroxisomes and Yeast Vacuoles - Section 12.3
Blue-Fluorescent and Nonfluorescent Probes
D1552
DAMP *
H22845
Hydroxystilbamidine †
L7525
LysoTracker Blue DND-22 †
L12490
LysoTracker Blue-White DPX
Green-Fluorescent Probe
L7526
LysoTracker Green DND-26
Yellow- and Orange-Fluorescent Probes
D113
Dansyl cadaverine
L12491
LysoTracker Yellow HCK-123
Red-Fluorescent Probes
L7528
LysoTracker Red DND-99
R14060
RedoxSensor Red CC-1 ‡
* Nonfluorescent probe. † Blue-fluorescent probe. ‡ The differential distribution of the oxidized product between mitochondria and
lysosomes appears to depend on the redox potential of the cytosol.
Probes for Acidic Organelles, Including Lysosomes (pH-Sensitive Probes) — Probes for Lysosomes, Peroxisomes and Yeast
Vacuoles - Section 12.3
Blue-Fluorescent Probes
L7533 LysoSensor Blue DND-167
L7545 LysoSensor Yellow/Blue DND-160 *
L22460LysoSensor Yellow/Blue 10,000 MW dextran *
Green-Fluorescent Probes
L7534 LysoSensor Green DND-153
L7535 LysoSensor Green DND-189
Yellow- and Orange-Fluorescent Probes
A1301 Acridine orange
L7545 LysoSensor Yellow/Blue DND-160 *
L22460LysoSensor Yellow/Blue 10,000 MW dextran *
Red-Fluorescent Probe
N3246 Neutral red
* Dual-emission spectrum.
Probes for the Endoplasmic Reticulum — Probes for the Endoplasmic Reticulum and Golgi Apparatus - Section 12.4
Blue-Fluorescent Probe
E12353 ER-Tracker Blue-White DPX
Green-Fluorescent Probes
B7447 BODIPY FL brefeldin A
D272 DiOC5(3)
D273 DiOC6(3)
Yellow- and Orange-Fluorescent Probes
B7449 BODIPY 558/568 brefeldin A
D282 DiIC18(3)
D384 DiIC16(3)
R648MPRhodamine B, hexyl ester
R634 Rhodamine 6G
T668
Tetramethylrhodamine, methyl ester
T669
Tetramethylrhodamine, ethyl ester
Probes for the Golgi Apparatus — Probes for the Endoplasmic Reticulum and Golgi Apparatus - Section 12.4
Nonfluorescent Probe
B7450 Brefeldin A
Green-Fluorescent Probes
B7447 BODIPY FL brefeldin A
D3521 BODIPY FL C5-ceramide *
B22650 BODIPY FL C5-ceramide complexed to BSA *
D3522 BODIPY FL C5-sphingomyelin
N1154 NBD C6-ceramide
N22651NBD C6-ceramide complexed to BSA
N3524 NBD C6-sphingomyelin
Orange-Fluorescent Probe
B7449 BODIPY 558/568 brefeldin A
Red-Fluorescent Probes
D3521 BODIPY FL C5-ceramide *
B22650 BODIPY FL C5-ceramide complexed to BSA *
D7540 BODIPY TR ceramide *
* Dual-emission spectrum.
Fluorescent Probes for Use with GFP - Note 12.1
Probes for Multiplexed Detection of GFP-Expressing Cells
The green-fluorescent protein (GFP) reporter has added a new dimension to the analysis of protein localization, allowing real-time
examination in living cells of processes that have conventionally been observed through immunocytochemical "snapshots" in fixed
specimens.
Using other spectrally distinct probes and markers (Table 1) adds extra data dimensions and reference points to
these experiments (Figure 1)
Figure 1. The morphology of sporulating Bacillus subtilis in the early stages of
forespore engulfment. The membranes and chromosomes of both the forespore and
the larger mother cell are stained with FM 4-64 (red; T3166, T13320) and DAPI (blue;
D1306, D3571, D21490), respectively. The small green-fluorescent patch indicates
the localization of a GFP fusion to SPoIIIE, a protein essential for translocation of the
forespore chromosome that may also regulate membrane fusion events (see Proc
Natl Acad Sci U S A 96, 14553 (1999)). The background contains sporangia at various
stages in the engulfment process stained with MitoTracker Green FM (green,
M7514) and FM 4-64 (red).
The majority of the applications summarized in Table 1 involve living cells, tissues and organisms. There are many other instances
where research objectives call for complementary use of immunochemical and GFP-based protein localization techniques. These
experiments demand the unmatched combination of brightness, photostability and spectral separation provided by our Alexa Fluor
dye–labeled secondary detection reagents. For two-color combinations with GFP, we recommend our Alexa Fluor 555, Alexa Fluor
568 or Alexa Fluor 594 dye–labeled secondary antibodies (Secondary Immunoreagents - Section 7.2, Summary of Molecular
Probes' secondary antibody conjugates - Table 7.1). For three-color detection, add Alexa Fluor 635 or Alexa Fluor 647 dye-labeled
antibodies. Some immunohistochemical procedures such as paraffin embedding of fixed tissue result in loss of the intrinsic
fluorescence of GFP. In other cases, GFP expression levels may simply be too low for detection above background
autofluorescence.
Antibodies to GFP provide remedies for these problems (Figure 2.. We offer unlabeled mouse monoclonal
and rabbit polyclonal antibodies to GFP (A6455, A11120, A11121, A11122; Primary Antibodies for Diverse Applications - Section
7.5) as well as Alexa Fluor dye–labeled rabbit polyclonal antibodies to GFP (A21311, A21312, A31851, A31852; Primary
Antibodies for Diverse Applications - Section 7.5).
Figure 2. HeLa cell transfected with pShooter pCMV/myc/mito/GFP, then fixed and
permeabilized. Green-fluorescent protein (GFP) localized in the mitochondria was labeled
with mouse IgG2a anti-GFP antibody (A11120) and detected with orange-fluorescent Alexa
Fluor 555 goat anti–mouse IgG antibody (A21422), which colocalized with the dim GFP
fluorescence. F-actin was labeled with green-fluorescent Alexa Fluor 488 phalloidin
(A12379), and the nucleus was stained with blue-fluorescent DAPI (D1306, D3571, D21490).
The sample was mounted using ProLong Gold antifade reagent (P36930). Some GFP
fluorescence is retained in the mitochondria after fixation (top), but immunolabeling and
detection greatly improve visualization (bottom).
Alexa Fluor Dyes — Highly Fluorescent FRET Acceptors
Proximity-dependent fluorescence resonance energy transfer (FRET) allows detection of protein–protein interactions with much
higher spatial resolution than conventional diffraction-limited microscopy.
Alexa Fluor dyes with strong absorption in the 500–
600 nm wavelength range are excellent FRET acceptors from GFP (Table 2). An assay to detect activation of GFP–GTPase fusions
developed by researchers at Scripps Research Institute
utilizes the GTPase-binding domain (PBD) of PAK1, a protein that binds
to GTPases only in their activated GTP-bound form. GTPase activation is indicated by FRET from GFP to PDB labeled with Alexa
Fluor 546 C5-maleimide at a single N-terminal cysteine residue. This assay has been used to determine the location and dynamics
of rac and Cdc42 GTPase activation in living cells.
Normalizing Expression and Translation Signals
In 2002, researchers in Scott Fraser's laboratory at the California Institute of Technology reported a method of coinjecting Texas
Red dye–labeled 10,000 MW dextran and GFP vectors into sea urchin embryos. This method overcomes a multitude of problems
inherent in making intra- and inter-embryo comparisons of gene expression levels using confocal microscopy. In particular, laser
excitation and fluorescence collection efficiencies vary with the depth of the fluorescent protein in the embryo, and the
orientation of different embryos on the coverslip varies relative to the microscope objective. Measuring the ratio of Texas Red
dextran and GFP fluorescence signals corrects for these spatial factors, providing a gene expression readout that is 2–50 times
more accurate than conventional confocal microscopy procedures depending on the localization of GFP within an embryo.
A
similar strategy was previously used to determine translation efficiencies of GFP-encoding mRNAs.
Table 1. Probes for multiplexed detection of GFP-expressing * cells.
Target
Probe
Cat #
Ex/Em
GFP Fusion Partner
Specimen
Reference
Physiological Indicators
Intracellular Ca2+ Fura-2 AM
F1201,
F1221,
F1225,
F14185
335/505 Protein kinase C (PKC)
BHK cells
Biochem J 337
( Pt 2), 211
(1999)
Intracellular Ca2+ X-Rhod-1 AM
X14210
580/602 Trpm5
580/602 (melastatin-related
cation channel)
CHO cells
Nat Neurosci
5, 1169 (2002)
Intracellular Ca2+ Fura Red AM
F3020,
F3021
GFP expressed
Mouse pancreatic
488/650 specifically in pancreatic
islets
β-cells
Human growth hormone RIN1046-38
(hGH)
insulinoma cells
Am J Physiol
Endocrinol
Metab 284,
E177 (2003)
Am J Physiol
Cell Physiol
283, C429
(2002)
Intracellular pH
5-(and 6-)Carboxy SNARF-1
C1271
AM ester acetate
568/635
Mitochondrial
membrane
potential
TMRM
T668
555/580 Cytochrome c
MCF-7 human breast J Cell Sci 116,
carcinoma, HeLa
525 (2003)
Superoxide (O2–) Dihydroethidium
D1168
518/605 Cytochrome c
J Biol Chem
MCF-7 human breast
278, 12645
carcinoma
(2003)
Synaptic activity FM 4-64
T3166,
T13320
506/750 §
VAMP (vesicle-associated Rat hippocampal
membrane protein)
neurons
Nat Neurosci
3, 445 (2000)
Rapsyn (receptoraggregating protein)
J Neurosci 21,
5439 (2001)
Receptors and Endocytosis
Acetylcholine
receptor
Tetramethylrhodamine αbungarotoxin
T1175
553/577
Epidermal
growth factor
(EGF)
Rhodamine EGF
E3481
555/581 EGF receptor
Endosomes
Transferrin from human
serum, Alexa Fluor 546
conjugate
T23364
556/573
Endosomes
Transferrin from human
serum, Alexa Fluor 568
conjugate
T23365
PrPc (cellular prion
578/603
protein)
β2-adrenergic receptor
(β2AR)
Zebrafish
Mol Biol Cell
MTLn3 rat mammary
11, 3873
adenocarcinoma
(2000)
HEK 293, rat
Brain Res 984,
hippocampal neurons 21 (2003)
SN56 cells
J Biol Chem
277, 33311
(2002)
Endosomes
FM 4-64
T3166,
T13320
506/750 §
PrPc (cellular prion
protein)
SN56 cells
J Biol Chem
277, 33311
(2002)
Organelles
Endoplasmic
reticulum
ER-Tracker Blue-White DPX E12353
375/520
HSD17B7 gene product
HeLa, NIH 3T3
(3-ketosteroid reductase)
Mol Endocrinol
17, 1715
(2003)
Golgi complex
BODIPY TR ceramide
589/617
PrPc (cellular prion
protein)
J Biol Chem
277, 33311
(2002)
Lysosomes
Mitochondria
Nuclear DNA
Nuclear DNA
D7540
SN56 cells
L7528
577/590 Heparanase
Primary human
fibroblasts, MDA-231 Exp Cell Res
(human breast
281, 50 (2002)
carcinoma)
MitoTracker Red
M7512
Sam5p (mitochondrial
578/599 carrier for Sadenosylmethionine)
Yeast (Saccharomyces EMBO J 22,
cerevisiae)
5975 (2003)
DAPI
D1306,
D3571,
D21490
358/461 Histone H2B
HeLa
Methods 29,
42 (2003)
Hoechst 33342
H1399,
H3570,
H21492
350/461 Histone H1
BALB/c 3T3 fibroblasts
Nature 408,
877 (2000)
HeLa
J Biol Chem
278, 33528
(2003)
LysoTracker Red
Nuclear DNA
SYTO 17
S7579
621/634 HIV-1 integrase
Nuclear DNA
SYTO 59
S11341
622/645
Nuclear DNA
TO-PRO-3
T3605
642/661 Citron kinase
HeLa
J Cell Sci 114,
3273 (2001)
DiI
D282,
D3911,
N22880
549/565 Synaptobrevin
Xenopus optic
neurons
Nat Neurosci
4, 1093 (2001)
Human peripheral
blood T cells (PBT)
Nat Immunol
5, 272 (2004)
NIH 3T3
J Cell Sci 113
Pt 21, 3725
(2000)
Plasma
membrane
Microtubule plus-end
binding protein
Porcine kidney
Mol Biol Cell
epithelial cells (LLCPK) 14, 916 (2003)
Other Subcellular Structures
ERM (ezrin-radixinmoesin) proteins
F-actin
Rhodamine phalloidin
R415
554/573
F-actin
Alexa Fluor 568 phalloidin
A12380
578/603 Calponin
Lipid rafts
Cholera toxin subunit B
(recombinant), Alexa Fluor C22842
594 conjugate
Histocompatibility
NK cell–B-cell
590/617 leukocyte antigen (HLA)- immunological
Cw4
synapse
Proc Natl Acad
Sci U S A 98,
14547 (2001)
GRASP65 (Golgi stacking
HeLa
protein)
J Cell Biol 156,
495 (2002)
Cell Classification Markers
Annexin V, Alexa Fluor 594
A13203
conjugate
590/617
Transformed B
lymphocytes
(Raji cells)
CellTracker Orange CMTMR C2927
ICAM-3 (intercellular
550/575
adhesion molecule-3)
T-lymphocytes and
Nat Immunol
antigen-presenting B
3, 159 (2002)
cells
Cell-surface
antigens
R-Phycoerythrin
(streptavidin conjugate)
S866,
S21388
565/575 GFP gene expression
NIH 3T3
Neurons
NeuroTrace 530/615 redfluorescent Nissl stain
N21482
Tau microtubule-binding
530/620 protein (Purkinje cell
Mouse brain slice
marker)
Neurons
Alexa Fluor 594 hydrazide
A10438,
A10442
588/613 Synaptophysin
Apoptotic cells
Cytometry 25,
211 (1996)
Aplysia californica
sensory neurons
J Neurosci 23,
6392 (2003)
Neuron 40,
151 (2003)
* This list covers only Aequoria victoria GFP, optimized mutants (e.g., EGFP) and green-fluorescent proteins from other species
(e.g., Renilla reniformis). Fluorescent proteins with distinctly different excitation and emission characteristics (CFP, YFP, dsRed,
etc.) are not included. Fluorescence excitation (Ex) and emission (Em) maxima, in nm. Simultaneous imaging of GFP with fura-2
or ER-Tracker Blue-White DPX requires excitation wavelength–switching capability, because the fluorescence emission spectra
overlap extensively. Even under these conditions, signal bleedthrough from one detection channel to the other may still be
problematic, depending on the expression level and localization of the GFP chimera. See Biochem J 356, 345 (2001) for further
discussion. § The fluorescence emission spectra of styryl dyes such as FM 1-43 and FM 4-64 are broad and extend into the green
emission range of GFP. In some cases, FM dye emission can overspill into the GFP detection channel, causing degraded resolution
of image features. The excitation and emission spectra of FM 1-43 overlap those of GFP more extensively than those of FM 4-64.
Therefore, using FM 4-64 instead of FM 1-43 is recommended to minimize this problem.
Table 2. R0 values for FRET from EGFP to Alexa Fluor dyes. *
Acceptor Dye
Alexa Fluor 546 dye
Alexa Fluor 555 dye
Alexa Fluor 568 dye
Alexa Fluor 594 dye
R0 (Å)
57
63
54
53
* R0 values in angstroms (Å) represent the distance at which fluorescence resonance energy transfer from the donor dye to the
acceptor dye is 50% efficient. Values were calculated from spectroscopic data as outlined (Fluorescence Resonance Energy
Transfer (FRET) - Note 1.2 ).