Titration curve of Caspase-7 activity assay using varying amounts of Jurkat cells, untreated or etoposide-treated (6 hours). Cell extracts were prepared for immunoprecipitation using Caspase-7 Antibody. Immune complexes were incubated with peptide substrate Ac-DEVD-AFC. Specific Caspase-7 activity was measured based on the fluorescent AFC group from the hydrolysis of Ac-DEVD-AFC by activated Caspase-7. Specific inhibition assay was performed by preincubating the immune complex with Ac-DEVD-CHO for 30 minutes before adding Ac-DEVD-AFC.
The Caspase-7 Activity Assay Kit includes all reagents necessary to assay caspase-7 activity, including Caspase-7 Antibody, fluorescent caspase substrate, reaction buffer and cell extract buffer. The assay system uses Caspase-7 Antibody to selectively immunoprecipitate caspase-7. Fluorogenic substrate is added and is cleaved proportionally to the amount of activated caspase-7, generating free fluorescent AFC. Free AFC is measured fluorometrically at lmax = 505 nM. Compared to other commercially available caspase-7 activity assays, this kit is designed to be more specific by using Caspase-7 Antibody to selectively immunoprecipitate caspase-7 from cell lysates.
Expected Molecular Weight: 35, 20 kDa
The kit components are designed to measure the endogenous caspase-7 enzymatic activity in human samples. Caspase-7 antibody immunoprecipitates human caspase-7 and is unique to this kit.
Caspase-7 (CMH-1, Mch3, ICE-LAP3) has been identified as a major contributor to the execution of apoptosis (1-4). Caspase-7, like caspase-3, is an effector caspase that is responsible for cleaving downstream substrates such as (ADP-ribose) polymerase and PARP (1,3). During apoptosis, caspase-7 is activated through proteolytic processing by upstream caspases at Asp23, Asp198, and Asp206 to produce the mature subunits (1,3). Similar to caspase-2 and -3, caspase-7 preferentially cleaves substrates following the recognition sequence DEVD (5).
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