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Ac-DEVD-AFC

Caspase-3 substrate
 
ALX-260-032-M001 1 mg 60.00 USD
 
ALX-260-032-M005 5 mg 173.00 USD
Do you need bulk/larger quantities?
 
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 Specification

Alternative Name:Caspase-3 substrate (fluorogenic)
 
Sequence:Ac-Asp-Glu-Val-Asp-AFC (AFC = 7-Amino-4-trifluoromethylcoumarin)
 
Formula:C30H34F3N5O13
 
MW:729.6
 
CAS:201608-14-2
 
Peptide Content:65-95%
 
Purity:≥96% (HPLC)
 
Appearance:White to off-white powder.
 
Solubility:Soluble in dimethyl formamide, DMSO or methanol; slightly soluble in water (0.4mg/ml).
 
Shipping:Ambient
 
Long Term Storage:-20°C
 
Handling:Protect from light. Keep cool and dry.
 
Technical Info/Product Notes:AFC has an excitation maximum of 400nm and an emission maximum of 505nm.
 
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.

 

Product Literature References

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;
Epidermal growth factor signaling protects from cholestatic liver injury and fibrosis: J. Svinka, et al.; J. Mol. Med. (Berl.) (2016), Application(s): Detection of active caspases, liver hepatocytes, Abstract; Full Text
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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Caspase substrate
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ALX-260-031-M001 1 mg 57.00 USD
 
ALX-260-031-M005 5 mg 144.00 USD
Do you need bulk/larger quantities?
 

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