Calpastatin is the endogenous intracellular inhibitor of calpains, calcium-dependent cysteine proteases. There are currently 14 distinct calpains in humans and rodents, with two different small subunit accessory proteins. By far the best studied are calpains 1 & 2, the ubiquitous calpains that differ in their sensitivity to calcium. Calpastatin inhibits calpain 1 & 2, but does not appear to inhibit calpain-3 (although there are conflicting reports in the literature). Other calpain family members (calpains 5-15) have more limited tissue distribution, and perhaps different functions, and little is known about the efficacy of calpastatin on these isoforms. A single gene for calpastatin is alternatively spliced into a seemingly endless variety of calpastatin isoforms, with different exons matched together, and differentially expressed in different tissues. Full-length calpastatin encodes a 708 amino acid protein with predicted mass of 76.484 kD, but modifications and cysteine-rich regions make the protein migrate at 110 kD on reduced SDS PAGE gels. The full-length calpastatin contain an aminoterminal "L" domain, followed by 4 repeats of the inhibitory domains. Different splice variants include the L domain with 1, 2 or 3 inhibitory domains, and the inhibitory domains can be expressed without the L domain. The inhibitory domains are further divided into subdomains A, B and C. The A and C subdomains are thought to enhance binding, and the B subdomains contain inhibitory activity. Post-translational modifications include phosphorylation, which modulates membrane binging. Calpastatin is also readily cleaved, separating the L domain from the inhibitory domain, and then further degraded to abolish activity. Sera from rheumatoid arthritis patients often contains inhibitory autoantibodies to calpastatin, which effectively increases the calpain activity. Autoantibodies to calpastatin have also been detected in infertile males, where it seems to compromise sperm activity. Calpastatin in expressed in virtually all cell types, severalfold in excess of the calpain concentration. RP2Calpastatin works well for Western Blotting applications, and when used against the reduced protein identifies bands at 110 Kd, 82 Kd, and a series of further cleaved active forms. RP2Calpastatin will also bind to the non-reduced protein, and thus has application for immunoprecipitation, immunohistochemistry, and ELISA. Most cell types produce Calpastatin; since it is a cellular protein, cell lysate works well for Western blots. Rat, mouse and human Calpastatin are detected with RP1Calpastatin. A recommended starting concentration for Western blots is 1:1000 when using colorimetric substrates such as BCIP/NBT, and 1:5000 for chemiluminescent substrates. Higher concentrations of antibody may be needed for samples from more distantly related species

FOR RESEARCH USE ONLY. NOT FOR USE IN HUMANS.

Triple Point Biologics products are sold exclusively through Abcam. For availability, pricing and buying information please select your country below and press 'Go'. You will be relocated to the relevant datasheet page on Abcam's website.