Caspase-5 Substrate WEHD-AFC

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Application
Enzyme Activity Assay (EAA), Functional Studies (Func)
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Sequence Ac-Trp-Glu-His-Asp-AFC
Characteristics Ready-to-use fluorometric substrate for caspase-1,-4,-5 and related caspases that recognize the amino acid sequence WEHD. Caspase-5 and related caspase activity can be quantified by fluorescent detection of free AFC after cleavage from the peptide substrate WEHD-AFC at Ex. = 400 nm and Em. = 505 nm, using a fluorometer or multi-well fluorescence plate reader. Alternatively, a shift in fluorescence from blue to green upon cleavage can be visualized using a hand-held long-UV lamp. The ready-to-use caspase substrate provides an economic alternative for researchers who perform large amount of caspase assays. Cell Lysis Buffer for caspase assays are also available separately.
Purity > 99 % by HPLC
Chemical Name Ac-WEHD-AFC, Caspase-5 Substrate, Fluorogenic
Formula C₃₈H₃₇F₃N₈O₁₁
Permeability Not-permeable
Molecular Weight 838.8 g/mol
Protocol 1. Induce apoptosis in cells by desired method. Concurrently incubate a control culture without induction.
2. Count cells and pellet 1-5 x 106 cells or use 50-200 µg cell lysates if protein concentration has been measured.
3. Resuspend cells in 50 µL of chilled Cell Lysis Buffer containing 10 mM DTT to each sample.
6. Add 5 µL of the 1 mM WEHD-AFC (50 µ M final conc.) into each tube individually and incubate at 37 °C for 1-2 hour.
7. Read samples in a fluorometer equipped with a 400-nm excitation filter and 505-nm emission filter. For a plate-reading set-up, transfer the samples to a 96-well plate. You may perform the entire assay directly in a 96-well plate. Fold-increase in caspase-5 activity can be determined by comparing these results with the level of the uninduced control.
Restrictions For Research Use only
Format Liquid
Handling Advice Protect from light and moisture
Storage -20 °C
Expiry Date 12 months
Product cited in: Liu, Shoji-Kawata, Sumpter, Wei, Ginet, Zhang, Posner, Tran, Green, Xavier, Shaw, Clarke, Puyal, Levine: "Autosis is a Na+,K+-ATPase-regulated form of cell death triggered by autophagy-inducing peptides, starvation, and hypoxia-ischemia." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 110, Issue 51, pp. 20364-71, 2013 (PubMed).

Bai, Goodrich: "Different DNA lesions trigger distinct cell death responses in HCT116 colon carcinoma cells." in: Molecular cancer therapeutics, Vol. 3, Issue 5, pp. 613-9, 2004 (PubMed).

Coletti, Yang, Marazzi, Sassoon: "TNFalpha inhibits skeletal myogenesis through a PW1-dependent pathway by recruitment of caspase pathways." in: The EMBO journal, Vol. 21, Issue 4, pp. 631-42, 2002 (PubMed).