Replaces Prod. #: BML-P409
Fluorogenic substrate for caspase-3 (CPP32), with a Km=9.7µM) and related cysteine proteases. Sequence is based on PARP cleavage at Asp216 for caspase-3. Similar to Ac-DEVD-AMC (Prod. No. ALX-260-031) but the AFC fluorophore has a greater Stokes’ shift upon cleavage than AMC. Reaction can be monitored quantitatively or visually using a hand-held long-UV lamp and visualizing a blue to green shift in fluorescence upon cleavage. Ex.: 400nm, Em.: 505nm.
Product Details
| Alternative Name: | Caspase-3 substrate (fluorogenic) |
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| Sequence: | Ac-Asp-Glu-Val-Asp-AFC (AFC = 7-Amino-4-trifluoromethylcoumarin) |
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| Formula: | C30H34F3N5O13 |
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| MW: | 729.6 |
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| CAS: | 201608-14-2 |
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| Peptide Content: | 65-95% |
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| Purity: | ≥96% (HPLC) |
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| Appearance: | White to off-white powder. |
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| Solubility: | Soluble in dimethyl formamide, DMSO or methanol; slightly soluble in water (0.4mg/ml). |
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| Shipping: | Ambient Temperature |
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| Long Term Storage: | -20°C |
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| Handling: | Protect from light. Keep cool and dry. |
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| Technical Info/Product Notes: | AFC has an excitation maximum of 400nm and an emission maximum of 505nm. |
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| Protocol: |
HEPES-Buffer (2x): 40 mM HEPES, pH 7.5, 20% glycerol, 4 mM DTT. Dilute to 1x with sterile distilled water prior to use.
Substrate: Prepare 20 mM stock solution in DMSO
- Induce apoptosis and prepare cell lysate or use recombinant caspase.
- Prepare reaction buffer: 10 µl of substrate stock solution + 1 ml 1x HEPES-Buffer for each reaction.
- Add an appropriate amount of cell lysate
(50-100 µl; should be titrated) or recombinant caspase to reaction buffer.
- Incubate for 1 hour at 37 °C.
- Measure with spectrofluorometer: 400 nm excitation wavelength, 505 nm emission wavelength.
- Suggested controls:
- Reaction mixture without substrate.
- Reaction mixture with non-apoptotic cell lysate.
- Reaction mixture with apoptotic cell lysate and caspase inhibitor. |
| |
| Regulatory Status: | RUO - Research Use Only |
| |
Product Literature References
Antitumoral Activity of Leptocarpha rivularis Flower Extracts against Gastric Cancer Cells: N. Carrasco, et al.; Int. J. Mol. Sci.
24, 1439 (2023),
Abstract;
Autophagy displays divergent roles during intermittent amino acid starvation and toxic stress-induced senescence in cultured skeletal muscle cells: D. Bloemberg & J. Quadilatero; J. Cell. Physiol.
236, 3099 (2021),
Application(s): Caspase-3 activity assay on cell lysates,
Abstract;
Toll-Like Receptor 2 Release by Macrophages: An Anti-inflammatory Program Induced by Glucocorticoids and Lipopolysaccharide: J. Hoppstadter, et al.; Front. Immunol.
10, 1634 (2019),
Abstract;
Full Text
Additive polyplexes to undertake siRNA therapy against CDC20 and survivin in breast cancer cells: M.B. Parmar, et al.; Biomacromolecules
19, 4193 (2018),
Abstract;
Epidermal growth factor signaling protects from cholestatic liver injury and fibrosis: J. Svinka, et al.; J. Mol. Med. (Berl.)
95, 109 (2017),
Application(s): Detection of active caspases, liver hepatocytes,
Abstract;
Full Text
Nimbolide reduces CD44 positive cell population and induces mitochondrial apoptosis in pancreatic cancer cells: S. Kumar, et al.; Cancer Lett.
413, 82 (2017),
Abstract;
Sub-lethal oxidative stress induces lysosome biogenesis via a lysosomal membrane permeabilization-cathepsin-caspase 3-transcription factor EB-dependent pathway: S.M. Leow, et al.; Oncotarget
8, 16170 (2017),
Abstract;
Full Text
Artesunate induces ROS-dependent apoptosis via a Bax-mediated intrinsic pathway in Huh-7 and Hep3B cells: Y. Pang, et al.; Exp. Cell Res.
16, 30161 (2016),
Application(s): Fluorometric assay for caspase-3 activity,
Abstract;
Decreased Poly(ADP-Ribose) Polymerase 1 Expression Attenuates Glucose Oxidase-Induced Damage in Rat Cochlear Marginal Strial Cells: Y. Zhang, et al.; Mol. Neurobiol.
53, 5971 (2016),
Abstract;
Mechanism of neem limonoids-induced cell death in cancer: Role of oxidative phosphorylation: N. Yadav, et al.; Free Radic. Biol. Med.
90, 261 (2016),
Application(s): Quantification of apoptosis and caspase activity measurement,
Abstract;
Organ specific alteration in caspase expression and STK3 proteolysis during the aging process: M. Lessard-Beaudoin, et al.; Neurobiol. Aging
47, 50 (2016),
Application(s): Caspase activity assays,
Abstract;
Full Text
Post-transcriptional control of executioner caspases by RNA-binding proteins: D. Subasic, et al.; Genes Dev.
30, 2213 (2016),
Application(s): Caspase activity reporter, HeLa Kyoto cells,
Abstract;
Full Text
The C-terminal domains of apoptotic BH3-only proteins mediate their insertion into distinct biological membranes: V. Andreu-Fernandez, et al.; J. Biol. Chem.
291, 25207 (2016),
Application(s): Cell culture to monitor caspase activity in cell extracts,
Abstract;
Full Text
Bothropoides pauloensis venom effects on isolated perfused kidney and cultured renal tubular epithelial cells: A.D. Marinho, et al. ; Toxicon
108, 126 (2015),
Application(s): Caspase activity in rat isolated kidney,
Abstract;
Elevation of soluble guanylate cyclase suppresses proliferation and survival of human breast cancer cells: H. C. Wen, et al.; PLoS One
10, e0125518 (2015),
Application(s): Cell Culture, Caspase 3 activity assay,
Abstract;
Full Text
Involvement of Bim in Photofrin-Mediated Photodynamically Induced Apoptosis: X. Wang, et al.; Cell Physiol. Biochem.
35, 1527 (2015),
Application(s): Cell Culture ,
Abstract;
Full Text
Oxidative phosphorylation-dependent regulation of cancer cell apoptosis in response to anticancer agents: N. Yadav, et al.; Cell Death Dis.
5, e1969 (2015),
Application(s): Cell culture, Fluorescence,
Abstract;
Full Text
Redox regulation of metabolic and signaling pathways by thioredoxin and GLUTAREDOXIn in nos-3 overexpressing hepatoblastoma cells: R. González, et al.; Redox Biol.
6, 122 (2015),
Application(s): Cell Culture, Fluorescence,
Abstract;
A novel TNFR1-triggered apoptosis pathway mediated by class IA PI3Ks in neutrophils: B. Geering, et al.; Blood
117, 5953 (2011),
Application(s): Caspase activity detected in human neutophils,
Abstract;
Full Text
Caspase-7 is activated during lovastatin-induced apoptosis of the prostate cancer cell line LNCaP: M. Marcelli, et al.; Cancer Res.
58, 76 (1998),
Abstract;
Different subcellular distribution of caspase-3 and caspase-7 following Fas-induced apoptosis in mouse liver: J.M. Chandler, et al.; J. Biol. Chem.
273, 10815 (1998),
Abstract;
Full Text
BAX-induced cell death may not require interleukin 1 β-converting enzyme-like proteases: J. Xiang, et al.; Proc. Natl. Acad. Sci. USA
93, 14559 (1996),
Abstract;
Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis: D.W. Nicholson et al.; Nature
376, 37 (1995),
Abstract;
Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE: Y. A. Lazebnik et al.; Nature
371, 346 (1994),
Abstract;
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