Caspase-5 Substrate WEHD-AFC

Application: Functional Studies (Func), Enzyme Activity Assay (EAA)

Catalog no.: ABIN412405

Additional Information

Immunogen: Sequence: Ac-Trp-Glu-His-Asp-AFC (AFC, 7-amino-4-trifluoromethyl coumarin)

Description: 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 (Cat. #1067-100, -400) and 2X Reaction Buffer (Cat. #1068-20, -80) for caspase assays are also available separately.

Molecular Weight: 952.0

Application Details

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 μ g cell lysates if protein concentration has been measured. 3. Resuspend cells in 50 μ l of chilled Cell Lysis Buffer (Cat.# 1067-100). 4. Incubate cells on ice for 10 minutes. 5. Add 50 μ l of 2X Reaction Buffer (Cat.# 1068-20, -80) containing 10 mM DTT (Cat.# 1201-1) to each sample. 6. Add 5 μ l of the 1 mM WEHD-AFC (50 μ M final conc.) into each tube individually and incubate at 37 o 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.

Purity: >99% by HPLC analysis.

Restrictions: For Research Use only

Publications

Product: Coletti, Yang, Marazzi et al.: "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).

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).