Calpain, Fluorometric BioAssay(TM) Kit

Activation of calpain is involved in many forms of physiological and pathological processes (e.g., apoptosis). Calpain activation requires cell membrane and Ca2, and activated calpain is released into cytosol. The Calpain Activity Assay Kit provides optimized buffers and reagents for a convenient measurement of calpain activity. The Extraction Buffer provided with the kit specifically extracts cytosolic proteins without contaminations of cell membrane and lysosome proteases. The Extraction Buffer also prevents auto-activation of calpain during the extraction procedure. Thus, the kit detects only activated calpain in cytosol upon treatment of cells with inducers (e.g., chemicals or drugs). The fluorometric assay is based on the detection of cleavage of calpain substrate Ac-LLY-AFC. Ac-LLY-AFC emits blue light (lmax=400nM); upon cleavage of the substrate by calpain, free AFC emits a yellow-green fluorescence (lmax=505nM), which can be quantified using a fluorometer or a fluorecence plate reader. Comparison of the fluorescence intensity from a treated sample with a normal control allows determination of the changes in calpain activity.\n\nSample Type:\nCell and tissue lysates\n\nIntended Use:\nThe kit provides a simple and convenient means for analyzing calpain activity in apoptotic and other samples. The assay is based on fluorometric detection of the cleavage of calpain substrate at 505nm using a fluorometer or a fluorecence plate reader.\n\nSpecificity:\nMammalian\n\nAbsorption: 400nm\n\nExcitement: 505nm\n\nFluorescence: Ex/Em 400/505nm \n\nKit components: \nExtraction Buffer 1x25ml\n10X Reaction Buffer 1x1.5ml\nCalpain Substrate Ac-LLY-AFC 1x500ul \nActive Calpain I (Positive Control) 1x10ul\nCalpain Inhibitor Z-LLY-FMK 1x10ul\n\nStorage and Stability:\nStore liquids at -70 degrees C. Store other components at 4 degrees C. Stable for 6 months For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.

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