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 |
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Application(s): Caspase-3 activity assay on cell lysates,
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Additive polyplexes to undertake siRNA therapy against CDC20 and survivin in breast cancer cells: M.B. Parmar, et al.; Biomacromolecules
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Epidermal growth factor signaling protects from cholestatic liver injury and fibrosis: J. Svinka, et al.; J. Mol. Med. (Berl.)
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Application(s): Detection of active caspases, liver hepatocytes,
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Nimbolide reduces CD44 positive cell population and induces mitochondrial apoptosis in pancreatic cancer cells: S. Kumar, et al.; Cancer Lett.
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Artesunate induces ROS-dependent apoptosis via a Bax-mediated intrinsic pathway in Huh-7 and Hep3B cells: Y. Pang, et al.; Exp. Cell Res.
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Decreased Poly(ADP-Ribose) Polymerase 1 Expression Attenuates Glucose Oxidase-Induced Damage in Rat Cochlear Marginal Strial Cells: Y. Zhang, et al.; Mol. Neurobiol.
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Application(s): Caspase activity reporter, HeLa Kyoto cells,
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Bothropoides pauloensis venom effects on isolated perfused kidney and cultured renal tubular epithelial cells: A.D. Marinho, et al. ; Toxicon
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Application(s): Caspase activity in rat isolated kidney,
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Elevation of soluble guanylate cyclase suppresses proliferation and survival of human breast cancer cells: H. C. Wen, et al.; PLoS One
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Application(s): Cell Culture, Caspase 3 activity assay,
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Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE: Y. A. Lazebnik et al.; Nature
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