Ac-DEVD-AFC

Caspase-3 substrate



ALX-260-032-M001	1 mg	49.00 USD

ALX-260-032-M005	5 mg	143.00 USD

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)

Formula:	C₃₀H₃₄F₃N₅O₁₃

MW:	729.6

Purity:	≥96% (HPLC)

Appearance:	White to off-white powder.

Sequence:	Ac-Asp-Glu-Val-Asp-AFC (AFC = 7-Amino-4-trifluoromethylcoumarin)

CAS:	201608-14-2

Peptide Content:	65-95%

Solubility:	Soluble in dimethyl formamide, DMSO or methanol; slightly soluble in water (0.4mg/ml).

Long Term Storage:	-20°C

Handling:	Keep cool and dry. Protect from light.

Background / Technical Information:	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

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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