Calpain Methods and Protocols by Valery F Thompson, Darrel E Goll (auth.), John S. Elce

By Valery F Thompson, Darrel E Goll (auth.), John S. Elce (eds.)

In Calpain equipment and Protocols, John S. Elce and a professional workforce of imperative investigators current a collection of confirmed and simply protocols for learning calpain. The tools contain in vitro ideas for the detection, expression, purification, and assay of µ- and m-calpain, supplemented with quite a lot of procedure and tissue versions 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 harm. one of the analytical concepts hired are casein zymography, immunofluorescence, and calpain job assays. The authors additionally study particular substrates which were proposed for the calpains. hugely useful and with no trouble repeatable, Calpain tools and Protocols deals investigators keen on easy and clinically orientated calpain learn a gold-standard choice of strong experimental instruments for locating the character and serve as of calpains.

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Elute the calpains with an appropriate linear gradient of NaCl concentration with a total volume of 40 mL. For μ-calpain the gradient should be from 0 to ~300 mM NaCl; for m-calpain the gradient should be from 0 to 500 mM NaCl. Under these conditions, μ-calpain is eluted at 185–200 mM NaCl and m-calpain at 380 mM NaCl (Figs. 2 and 3). Calpastatin (always applied separately) elutes at ~220 mM NaCl (2). 5. Collect fractions of 1 mL and assay aliquots for proteolytic activity (see Note 6). 6. For Western blotting, the enzyme-containing fractions can be desalted on a small Sephadex G-25 column (PD-10, Pharmacia) according to the manufacturer’s instructions.

Add the enzyme sample to the casein resin and mix well for 2–5 min (see Note 16). 4. Transfer the mixture to an appropriately sized column and wash out unbound proteins with casein/Ca2+ buffer. 5. 5, 5 mM EGTA, 2 mM EDTA, 5 mM 2-ME. 6. Dialyze the eluted enzyme extensively against an appropriate buffer to remove excess chelator and Ca2+-chelator complexes, and casein fragments, especially if active enzyme has been purified. The use of an immobilized peptide from the Ca2+-ATPase for affinity purification of μ-calpain (8) is described in Chapter 5.

Biochem. 143, 30–34. Affinity Chromatography 33 4 Purification of Calpain by Affinity Chromatography on Reactive Red-Agarose or on Casein-Sepharose Dorothy E. Croall 1. Introduction There is no single affinity chromatography method that is universally effective for the purification of calpains. Dye affinity chromatography and calcium-dependent binding to immobilized substrates or inhibitors are the most commonly attempted affinity methods. Our laboratory has used Reactive Red-agarose in the purification of m-and μ-calpain from bovine heart and other tissues for many years (1,2).

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