Calpain proteinases are defined currently as papain-like neutral proteases that are calcium activated. Calpains 1 and 2 are composed of a large subunit, which is proteolytically active, and a small subunit (also called calpain-4), or enhancer protein, that is not proteolytically active. The other calpain proteins identified to date are not known to require a small subunit, although there is evidence that the calpain small subunit #2 may act as a chaperonin in the folding of calpains, and then disassociate. Domains in the large subunit include the amino terminal domain-I, the proteinase domain-II, domain-III, and the EF-hand domain-IV (Domain-T in calpains 5 & 6). Calpain-8, also known as NCL-2 (novelcalpain-2), was originally characterized in rodents as a stomach specific protein, but the human sequence was isolated from leukocytes. Most similar to calpain-2 (60 % identical to rat or human sequences), murine calpain-8 is also known as a truncated form (Calpain NCL-2'), lacking the calcium binding domain, and most of domain-III. Homology between human calpain-8 and human calpain-9 (the human stomach-specific calpain) is much lower; calpain-9 is more similar to calpain-3. Homology between rat, mouse and human calpain-8 is high, and RP2Calpain-8 recognizes all three species of protein. RP2Calpain-8 recognizes both the long and short (NCL-2') forms of calpain-8. The large subunit of calpain-8 zymogen runs at approximately 80 Kd, and the amino terminal truncation at activation yields an approximately 60 kD form. Cleavage of the carboxyterminal region generates smaller forms of Calpain-8, but it is not clear if these forms are proteolytically active. RP2Calpain-8 works well for Western Blotting applications, and when used against the reduced protein identifies bands at 80 kD, 60 Kd, and a series of further cleaved forms. RP2Calpain-8 will also bind to the non-reduced protein, and thus has application for immunoprecipitation, immunohistochemistry, and ELISA. 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

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