Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
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) P.1 FluoProbes® 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 P.2 FluoProbes® FT-27060A 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 - - - 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) P.3 FluoProbes® FT-27060A 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 P.4 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. Rev.S03E-F02VB P.5