Download FluoProbes Luciferin substrates

FluoProbes®
FT-27060A
Luciferins
Products Information
Product name
cat.numbe
MW
(g·mol-1)
D-Luciferin, free acid
FP-27060A, 10 mg
FP-270609, 25 mg
FP-27060B, 250 mg
FP-27060D, 1 g
D-Luciferin, Na salt
FP-726045, 10mg
FP-72604A, 25 mg
FP-72604B, 50 mg
FP-72604C, 250mg
FP-72604D, 500mg
D-Luciferin, K salt
FP-M1224A, 25 mg
FP-M1224B, 50 mg
FP-M1224C, 500 mg
FP-M1224D, 1 g
L-Luciferin, K salt
FP-U54040, 10 mg
Storage:
exc\em. exc\em.
(nm)
(nm)
mol. abs. Solubility Comments
(M-1cm-1)
280.33 328 / 532
18 000
DMSO Make your buffers alkaline prior to
Not soluble dissolving the D-Luciferin Free Acid, then
in neutral titrate to your desired pH.
buffers CAS: 2591-17-5
302.31 328 / 533
17 000
water pH>6
CAS: 103404-75-7
Recommended salt form for in vivo
318.42 328 / 533
17 000
water pH>6
CAS: 115144-35-9
318.42
–20°C >1 year. (M)
Protect from light and moisture. Long term storage at –70°C
Introduction
The highly purified synthetic luciferin exhibits physical properties identical to those of natural luciferin, isolated
from fireflies (Photinus pyralis) and other beetles. Enzymatic activity is optimal.
Luciferin is a generic term for photon emitting biomolecules, well represented in several marine species,
bacteria, protistes, fishs, insects r.
D-Luciferin is a luminescent substrate for firefly luciferase, a monomeric 61kDa protein (encoded by luc gene),
generating a green light flash. It is primarily used in reporter assays and ATP assays. Its bioluminescent reaction
is the most efficient known in nature! (With about 90 % of the energy released being converted to light): ATPdependent oxidation of luciferin by luciferase results in bioluminescence (Em = 560 nm) that is longer and
brighter in presence of CoenzymeA, at neutral and alkaline pH. Bioluminescence is red-shifted (Em = 617 nm)
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FT-27060A
under acidic conditions. Optimized formulations allow linear results over >8 orders of magnitude of enzyme
concentration, down less than 10–20 moles of luciferase.
The luciferin/luciferase system is used as a very sensitive reporter assay for gene expression in plants, bacteria,
mammalian cells, and for monitoring baculovirus gene expression in insects. It can be used too for ATP assays in
research applications or for detect bacterial contamination detection (used to measure 10 -15 molar quantity of
ATP). It also has been used for detecting certain amphipathic and hydrophobic substances, including anesthetics
and hormones, as these compete with luciferin for the hydrophobic site on the luciferase molecule (Anal
Biochem 190, 304 (1990)).
Production of light can be monitored with either a luminometer, including multiwell plate automatized
instruments, or a scintillation counter.
Directions for use
Handling and Storage
Luciferin free acid is soluble in DMSO at pH>6 but may be dissolved directly in Tricine or HEPES buffer (max.
1.5~mM). Potassium salt and Sodium salt are soluble in water or aqueous buffer up to 100mM at pH>6.
Protocol 1: Luciferin Reporter Assay
1- Dissolve 1 mM luciferin or luciferin salt, 3 mM ATP, Triton-X 100 (1%), 25 mM glycylglycine, PH 7.8, 15
mM MgSO4 , 4 mm EGTA and 1 mM DTT in fresh desionized ATP free water
Note: The Luciferin concentration can be checked by absorbance measurement at 385nm in 0.5 M
carbonate buffer, pH 11.5. Molar extinction is 18 000 M-1cm-1.
2- Warm luciferin substrate reagent to room temperature before starting.
3- Lyse cells using luciferin substrate reagent.
4- Pipet 5-10 µl of cell lysate into a microplate. Use buffer without cells as blank.
5- Prime luminometer with luciferin substrate solution according to manufacturer’s instructions.
6- Set luminometer to inject 200 µl of Luciferin Substrate with no delay and a 10 -second integration time.
Notes:
• The numerical instrument results (RLUs) for a given sample or standard will vary from day to day. However,
the relative differences between samples or standards should be consistent.
• If testing for ATP minimize all possible sources of ATP contamination by wearing gloves and using only ATPfree containers. Use only sterile ATP-free water and reagents (autoclave water and use autoclaved water for all
reagent prep).
• Sanitize luminometer injector lines each day before running samples. Use 1% bleach or other sterilant. Ask
luminometer manufacturer for appropriate solution for their instrument.
• Store any substrate or samples containing ATP in polypropylene or glass only. Avoid polystyrene.
• Purified luciferase may be used as a positive control.
Here are proven buffer systems that are known maximize performance and sensitivity:
Notes: To dissolve in buffered saline start by using the predicted molar alkalinity using Sodium Hydroxide
(NaOH) added to the buffer before adding d-Luciferin. If a precipitate forms adjust the pH higher until the
solution is clear.
Protocol 2: Assay-Reagents for Firefly Glow Luminescence
20 mM Tricine
1.07 mM (MgC03)4 . Mg(OH)2 . 5H20
2.67 mM MgSO4
0.1 mM Ethylenediamine tetra-acetic acid (EDTA)
33.3 mM Dithiothreitol (DTT)
270 µM Coenzyme A
470 µM D-Luciferin
530 µM Adenosine Triphosphate (ATP)
* Adjust pH to 7.8 Protect from light, may store at –70º C
Protocol 3: Assay-Buffer for the measurement of ATP using Firefly Luciferase
300 µM D-Luciferin
5 µg protein/ml Firefly Luciferase
75 µM Dithiothreitol (DTT)
25 mM HEPES
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6.25 mM MgCl2
0.63 mM Ethylenediamine tetra-acetic acid (EDTA)
1.0 mg Protein/ml Bovine Serum Albumin (BSA)
*Adjust to pH 7.8 Protect from light, may store at –70º C
Apply Firefly Luciferase immediately before measurement starts
All liquids that used for in vivo tests and experiments, we recommended make a sterile filtration,
please filtrate through 0.25 µm filter.
Other protocol may found in the literature.
References
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-
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Alam, J., et al., Anal. Biochem. 188, 245 (1990).
Baracca A, e al., "Catalytic activities of mitochondrial ATP synthase in patients with mitochondrial DNA T8993G
mutation in the ATPase 6 gene encoding subunit a.", J Biol Chem, 275, 4177 (2000)
Brasier, A., et.al., “Optimized use of the Firefly Luciferase assay as a reporter gene in mammalian cell lines.”,
Biotechniques, 7, 1116 (1989).
Bronstein I., et.al., “Chemiluminescent and Bioluminescent reporter gene assays (Review).”, Anal. Biochem, 219, 169
(1994).
Creton R, et al., "Chemiluminescence microscopy as a tool in biomedical research.", Biotechniques, 31, 1098 (2001)
de la Monte SM, et al., "ATP luminescence-based motility-invasion assay.", Biotechniques, 33, 98 (2002)
DeLuca M., et al. “Firefly Luciferase: Mechanism of action, cloning and expression of the active enzyme.”, J. Biolum.
Chemilum. 3, 1(1989).
DeLuca, et al., “Two kinetically distinguishable ATP sites in firefly luciferase.”, Biochem. Biophys. Res. Commun, 23,
764 (1984).
Dyer B. et al., A noncommercial dual luciferase enzyme assay system for reporter gene analysis, Anal Biochem.
282(1):158-61 (2000) Abstract (Dual Luciferase Assay)
Frampas E. et al., The intraportal injection model for liver metastasis: advantages of associated bioluminescence to
assess tumor growth and influences on tumor uptake of radiolabeled anti-carcinoembryonic antigen antibody, Nuclear
Medicine Communications: Volume 32, Issue 2, p 147–154 (2011) Abstract
Gampala SS, et al., « Functional interactions of lanthanum and phospholipase D with the abscisic acid signaling
effectors VP1 and ABI1-1 in rice protoplasts”, J Biol Chem , 276, 9855 (2001)
Gould, S.J, et al., “Firefly Luciferase as a tool in molecular and cell biology.”, Anal. Biochem., 175, 5 (1988)
Hampf M. et al., A protocol for recombined Photinus and Renilla luciferase quantification compatible with protein
assays, Analytical Biochemistry 356:94-99 (2006) Abstract (Dual Luciferase assay)
Logeart-Avramoglou D. et al., In vitro and in vivo bioluminescent quantification of viable stem cells in engineered
constructs, Tissue Eng Part C Methods 16(3): 447-458 (2010) Abstract
Logeart-Avramoglou D. et al., An assay for the determination of biologically active bone morphogenetic proteins
using cells transfected with an inhibitor of differentiation promoter-luciferase construct, Analytical Biochemistry 349,
Issue 1:78-86 (2006) Abstract
Mackenzie GG, et al., "Low intracellular zinc impairs the translocation of activated NF-kappa B to the nuclei in
human neuroblastoma IMR-32 cells”, J Biol Chem, 277, 34610 (2002)
Manassero M. et al., Comparison of Survival and Osteogenic Ability of Human Mesenchymal Stem Cells in
Orthotopic and Ectopic Sites in Mice, Tissue Engineering Part A., Vol. 22, No. 5-6: 534-544 (2016) Abstract
Oudina K. et al., Noninvasive Bioluminescent Quantification of Viable Stem Cells in Engineered Constructs, Methods
in Molecular Biology, Volume 740, 165-178 (2011) Abstract
Rousseau J. et al., Advantages of bioluminescence imaging to follow siRNA or chemotherapeutic treatments in
osteosarcoma preclinical models, Cancer Gene Therapy 17, 387–397 (2010) Article
Sorensen CE, et al., "Visualization of ATP release in pancreatic acini in response to cholinergic stimulus. Use of
fluorescent probes and confocal microscopy." J Biol Chem, 276, 32925 (2001)
Webster J.J, et al., “Optimization of the Firefly Luciferase Assay for ATP.”, J. Appl. Biochem, 2, 1469 (1980).
White EH, et al., "Amino Analogs of Firefly Luciferin and Biological Activity Thereof.", J Am Chem Soc, 88, 2015
(1966)
Yang NC, et al., "A convenient one-step extraction of cellular ATP using boiling water for the luciferin-luciferase assay
of ATP”, Anal Biochem , 306, 323 (2002)
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FluoProbes®
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Modified luciferins
D-Luciferin-6-O-β-Dgalactopyranoside
FP-CQ6410 (M)
CAS: 131474-38-9
MW: 442.47
D-Luciferin, Phosphate
FP-DT244A, ,1mg
CAS: 145613-12-3
MW: 426.25
A/E: 328 / 533
Soluble in Water
Tech Sheet
D-Luciferin phosphate is a highly sensitive
bioluminescent substrate for detecting alkaline
phosphatases and protein phosphatases by
luminometric detection. It is used in combination
with luciferase/ATP system. D-Luciferin phosphate
can be converted to D-Luciferin by phosphatases (such as alkaline phosphatases, acid
phosphatases and protein phosphatases) that gives luminescence in the presence of luciferase and
its cofactors. Our Amplite luciferase assay kit can be used for detecting D-luciferin release from
D-Luciferin phosphate by phosphatases.
•Amino Luciferins
6-Amino-D-luciferin
6-Amino-6-deoxyluciferin
FP-JQ6990
CAS: 118969-27-0
MW : 279.34
(M)
N-CBZ-β-glycinyl-6aminoluciferin
FP-LV6220 (x)
CAS: 1233518-28-9
MW: 470.53
Soluble in H2O (pH > 8), DMSO
6-FMOC-Amino-D-luciferin
FP-EZD550 (X)
CAS: 578705-74-2
MW :501.59
Benzoyl-Leu-Gly-ArgAminoluciferin
FP-GDA700, 1mg (M)
CAS:73918-26-0
MW: 823.86
Soluble in DMSO
A/E: none/560nm
Tech Sheet
used for developing luminescent protease substrates
used as a reference standard for the luminescence assays
a luminogenic substrate for
complement component C3/C5
convertases, coagulation factor Xa,
and soybean trypsin-like enzyme. It is
also hydrolyzed by macropain, a high
molecular weight thiol proteinase from
human erythrocytes. The proteasereleased D-aminoluciferin can be
detected using our luciferase assay kits. It is much more sensitive than Boc-Leu-Gly-Arg-AMC,
the widely used fluorogenic AMC substrate.
Harwood KR et al(2011)
Identification of mutant firefly luciferases that efficiently utilize aminoluciferins. Chem Biol, 18, 1649.
Takakura H et al (2011)
Aminoluciferins as functional bioluminogenic substrates of firefly luciferase. Chem Asian J, 6, 1800.
•Inhibited luciferins/IIr substrates
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FluoProbes®
FT-27060A
D-Luciferin, ethyl ester
FP-CF4421, 10 mg
CAS: 135251-85-3
MW: 308.38
D-Luciferin, methyl ester
FP-UVC630, 5mg
CAS:73918-26-0
MW:294.3
These D-Luciferin analogs contain a ethyl or methyl moiety attached at the carboxy position, thus
preventing it be recognized by the firefly luciferase enzyme. However, the ester is hydrolyzed
effectively by lipase activity, and the resulted D-luciferin is well recognized by luciferase. DLuciferin methyl ester is a sensitive substrate for the chemiluminescent measurement of lipase
activity in homogeneous assays, or in cell lysate samples in combination with luciferase and its
cofactors. Soluble in DMSO
References
C3-Luc Cells Are an Excellent Model for Evaluation of Cellular Immunity following HPV16L1 Vaccination
Li-Li Li et al; Journal: PloS one (2016): e0149748
Genome-wide microRNA analysis identifies miR-188-3p as novel prognostic marker and molecular factor
involved in colorectal carcinogenesis
Martin Pichler; Journal: American Association for Cancer Research (2016): clincanres--0497
Identification of a Novel Protein Kinase A Inhibitor by Bioluminescence-Based Screening
Authors: Tetsuya Ishimoteto al; Journal: Biological and Pharmaceutical Bulletin (2015): 1969--1974
Discovery of novel adenylyl cyclase inhibitor by cell-based screening
Hiroki Mano et al; Journal: Biological and Pharmaceutical Bulletin (2014): 1689—1693
D-Luciferin, 6'methyl ester
FP-APIRO0, 10mg
CAS: 24404-90-8
MW: 294.35
Inhibitor of the firefly luciferase light producing system. This luciferin analog is useful in
chemiluminescent dual assay systems with both luciferase and cytochrome P450 enzymes.
Dealkylase removes the methyl ether which can then react with luciferase to produce light, in
direct correlation with the P450 enzyme levels and activity.
D-Luciferin, 6'-methyl ester, Na
FP-M1418B, 100 mg
CAS: 3022-11-5 (Na salt)
MW: 316.33
D-Luciferin, Acetate
FP-UVC640, 5 mg (M)
MW:322.36
MW: 316.33
D-Luciferin-AM
FP-M1909A (M)
CAS:73918-26-0
MW: 352.39
D-Luciferin acetate contains an acetate moiety attached at the 6-O-position, thus preventing it be
recognized by the firefly luciferase enzyme. However, the acetate moiety is removed effectively
by esterase activity, and the resulted D-luciferin is well recognized by luciferase. D-Luciferin
acetate is a sensitive substrate for the chemiluminescent measurement of esterase activity in
homogeneous assays, or in cell lysate samples in combination with luciferase and its cofactors.
Soluble in DMSO
D-Luciferin-AM ester contains an acetoxymethyl moiety attached at the carboxyl, thus preventing
it be recognized by the firefly luciferase enzyme. However, the AM ester moiety is removed
effectively by esterase activity, and the resulted D-luciferin is well recognized by luciferase.
Tech Sheet
Related products
·
·
·
·
Firefly Luciferase, recombinant, FP-D1826B
ATP disodium salt, 00064A
Luciferase reporter gene assay kit, FP-JQ6810
DMNPE-caged luciferin (cross easily
biological membranes), FP-21639A
·
·
·
·
Firefly & Renilla Luciferase Single Tube Assay
kit (dual luciferase), BE7812
Coelenterazine native, UP972333
BTA-2 (Dual luciferase assay), FP-QU6360
Coenzyme A, 627374
Ordering information
Catalog size quantities and prices may be found at http://www.fluoprobes.com
Please inquire for higher quantities (availability, shipment conditions).
For any information, please ask : FluoProbes® Hotline : +33(0)4 70 03 73 06
Disclaimer : Materials from FluoProbes® are sold for research use only, and are not intended for food, drug, household, or cosmetic use.
FluoProbes® is not liable for any damage resulting from handling or contact with this product.
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