By Valery F Thompson, Darrel E Goll (auth.), John S. Elce (eds.)
In Calpain equipment and Protocols, John S. Elce and a pro group of primary investigators current a suite of confirmed and simply protocols for learning calpain. The tools comprise in vitro thoughts for the detection, expression, purification, and assay of µ- and m-calpain, supplemented with a variety of approach and tissue types for learning either the physiological services and the results of inhibitors on calpain. The platforms used comprise neural tissue, kidney, liver, the attention, and membrane fusion in muscle and erythrocytes, every one in reference to hypoxia or different damage. one of the analytical concepts hired are casein zymography, immunofluorescence, and calpain job assays. The authors additionally learn particular substrates which have been proposed for the calpains. hugely useful and with no trouble repeatable, Calpain equipment and Protocols deals investigators enthusiastic about easy and clinically orientated calpain learn a gold-standard number of robust experimental instruments for locating the character and serve as of calpains.
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In Calpain tools and Protocols, John S. Elce and a professional group of significant investigators current a collection of confirmed and simply protocols for learning calpain. The equipment comprise in vitro options for the detection, expression, purification, and assay of µ- and m-calpain, supplemented with quite a lot of procedure and tissue types for learning either the physiological features and the results of inhibitors on calpain.
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0, 1 M NaCl, 1 mM EDTA, 1 mM EGTA, 5 mM 2-ME. Activity was measured by hydrolysis of 14C-labeled casein and the results are plotted as acid-soluble cpm of 14C-released in 15 min (A) or 30 min (B) at 25°C. 1. Preparation of Casein-Sepharose Couple casein to commercially available CNBr-Sepharose using the conditions recommended by the manufacturer. 1. Soak and prewash dry resin (normally 1–2 g) with 1 mM HCl, to give a resin bed volume of 5–10 mL. 2. 2) and mix immediately with 2 vol of 20 mg/mL casein in coupling buffer.
14. , Hammond, P. , Hartwell, R. , Scawen, M. , Sherwood, R. , Small, D. , Bruton, C. , Harvey, M. , and Lowe, C. R. (1981) Triazine-dye affinity chromatography. Biochem. Soc. Trans. 9, 290–293. 15. Stellwagen, E. (1990) Chromatography on immobilized reactive dyes. Methods Enzymol. 182, 343–357. 16. Reville, W. , Goll, D. , Stromer, M. , Robson, R. , and Dayton, W. R. 1976. A Ca2+- activated protease possibly involved in myofibrillar protein turnover: Subcellular localization of the protease in porcine skeletal muscle.
Thompson, V. , Goll, D. , and Antin, P. (1998) The bovine calpastatin gene promoter and a new N-terminal region of the protein are targets for cAMP-dependent protein kinase activity. J. Biol. Chem. 273, 660–666. 10. Geesink, G. , and Koohmaraie, M. (1998) An improved purification protocol for heart and skeletal muscle calpastatin reveals two isoforms resulting from alternative splicing. Arch. Biochem. Biophys. 356, 19–24. 11. Thompson, V. , Goll, D. , and Kleese, W. C. (1990) Effects of autolysis on the catalytic properties of the calpains.