Use

Calpain-94, also known as Calpian-3 or Muscle Calpain, is an intracellular, calcium-dependent cysteine protease. The calpains have papain-like activity, thus the –pain nomenclature. The calpain family members consist of a common small subunit, and a large variable subunit. Domains in the large subunit include the amino terminal domain-I, the proteinase domain-II, domain-III, and the EF-hand domain-IV. Unlike M-calpain and m-calpain, which are ubiquitously expressed, Calpain-94 is a member of the tissue-specific Calpains. Also, the endogenous calpain inhibitor, calpastatin, does not seem to inhibit Calpian-94. The half-life of native Calpain-94 is also much shorter; about 10 minutes. Mutations in Calpain-94 have been linked to Limb-Girdle Muscular Dystrophy, and inhibition of cysteine proteases has been shown to reduce symptoms of muscular dystrophy. Splice variants in calpain-3 yield a number of different isoforms (LP82, 85, 88, 90), which have a different aminoterminus, and different exons spliced in or deleted. The RP5Calpain-3 epitope follows the autocleavage site in insert #1, and thus represents the aminoterminus of cleaved calpain-3. RP5Calpain-3 recognizes all of the calpain-3 isoforms. RP5Calpain-3 recognizes latent and active Calpain-94. The latent large subunit is 94 kD, and the amino terminal truncation at activation yields an approximately 64- 62 Kd form. A cascade of smaller forms can be seen with further activation. RP5Calpain-3 works well for Western Blotting applications, and when used against the reduced protein identifies bands at 94 Kd, 82 Kd, 62 Kd, 60 Kd, and a series of further cleaved active forms. RP1Calpain-94 will also bind to the non-reduced protein, and thus has application for immunoprecipitation, immunohistochemistry, and ELISA. Calpain-94 is a cellular protein, thus cell lysates work well for Western blots. 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. EDTA/EGTA treatment of tissues or lysates is required to see latent zymogen.

FOR RESEARCH USE ONLY. NOT FOR USE IN HUMANS.

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