Caspase-1 Substrate YVAD-AFC

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

Catalog no.: ABIN412408

Additional Information

Immunogen: Sequence: Ac-Tyr-Val-Ala-Asp-AFC (AFC, 7-amino-4-trifluoromethyl coumarin)

Description: Ready-to-use fluorometric substrate for caspase-1/ICE and related caspases that recognize the amino acid sequence YVAD. Caspase-1 and related caspase activity can be quantified by fluorescent detection of free AFC after cleaved from the peptide substrate YVAD-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 volume caspase assays. Cell Lysis Buffer (Cat. #1067-100, -400) and 2X Reaction Buffer (Cat. #1068-20, -80), and DTT used for caspase assays are also available separately.

Molecular Weight: 719.7

Application Details

Protocol: 1. Induce apoptosis or treat cells by desired method. Concurrently incubate a control culture without treatment. Note: Active recombinant human caspase-1 is available to use as a positive control (BioVision, Cat.# 1081-25, -100). 2. Pellet 2-5 x 106 cells or use 100-300 μg cell lysates if protein concentration has been measured. 4. Resuspend cells in 50 μl of chilled Cell Lysis Buffer (Cat.# 1067-100). 5. Incubate cells on ice for 10 minutes. 6. Add 50 μl of 2X Reaction Buffer (Cat.# 1068-20,-80) containing 10 mM DTT (Cat.# 1201- 1) to each sample. 6. Add 5 μl of the 1 mM YVAD-AFC substrate (50 μ M final concentration) and incubate at 37 o C for 1-2 hour. 7. Read samples in a fluorometer equipped with a 400-nm excitation and 505-nm emission filters. For a plate-reading set-up, transfer the samples to a 96-well plate. You may also perform the entire assay directly in a 96-well plate. Fold-increase in YVAD-dependent caspase activity can be determined by comparing these results with the level of the untreated control .

Purity: >98% 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).

Ray, Akbiyik, Bernstein et al.: "CD40 engagement prevents peroxisome proliferator-activated receptor gamma agonist-induced apoptosis of B lymphocytes and B lymphoma cells by an NF-kappaB-dependent mechanism." in: Journal of immunology (Baltimore, Md. : 1950), Vol. 174, Issue 7, pp. 4060-9, 2005 (PubMed).