Use

Both M-calpain and ?-calpain are ubiquitously expressed in mammalian cells, and are countered by the endogenous calpain inhibitor, calpastatin. Other calpain family members (calpain-3, calpain-5, calpain-9, etc) have more limited tissue distribution, and perhaps different functions. The calpain family members consist of a common small subunit (Calpain-S1), and a large variable subunit. It is not clear that all calpains form heterodimers with a small subunit, but calpain-S1 is known to form a heterodimer with calpain-1 and also with calpain-2. Calpain S2 was recently discovered, and initial studies indicate that both small subunits may act as chaperonins to help with folding, or accessory proteins to help localize the calpains. Domains in the small subunit include the amino terminal domain-V (a glycine-rich sequence that is removed on activation of calpain, shorter in calpain-S2 than in calpain S1) and domain-VI, the calcium binding, EF hand domain. RP3Calpain-S2 recognizes latent and amino-processed calpain S1; the latent small subunit is 30 Kd, and the amino terminal truncation at activation yields an approximately 28 kD form. RP3Calpain-S2 Anti-Calpain S2 works well for Western Blotting applications, and when used against the reduced protein identifies bands at 28 kD and 26 kD. RP3Calpain-S2 will also bind to the non-reduced protein, and thus has application for immunoprecipitation, immunohistochemistry, and ELISA. Most cell types produce Calpain S1, but the distribution of calpain S-2 in unknown; since it is a cellular protein, cell lysate works well for Western blots. Calpain S2 is quickly degraded, and samples must be lysed in the presence of protease inhibiters to maintain latency. Rat, mouse and human Calpain S2 are detected with RP3Calpain-S2. 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.

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