Casein zymography is used to detect calpain activity in cell or tissue lysates. In this technique, lysates are loaded into a polyacrylamide gel containing casein, and the enzymes within the lysates are electrophoretically separated. The casein embedded in the gel acts as a substrate for calpains in the lysates, and its degradation reflects the activity of these enzymes. Calpain degradation of the casein is visualized as distinct bands that are devoid of dye when stained with Coomassie Brilliant Blue G-250. We describe here how calpains can be extracted from muscle tissue and assayed for activity using this technique. This technique is also generally applicable to lysates from other types of cells or tissues.
Biswas AK, Tandon S, Sharma D (2016) Identification and characterization of different domains of calpain and their influence on post-mortem ageing of goat meat during holding at 4±1 °C. LWT-Food Sci Technol 71:60–68CrossRefGoogle Scholar
Raser KJ, Posner A, Wang KK (1995) Casein zymography: A method to study μ-Calpain, m-calpain and their inhibitory agents. Arch Biochem Biophys 319:211–216CrossRefGoogle Scholar
James GT (1978) Inactivation of the protease inhibitor phenylmethylsulfonyl fluoride in buffers. Anal Biochem 86(2):574–579CrossRefGoogle Scholar
Beynon RAYJ, Bond JS (1989) Proteolytic enzymes: a practical approach. IRL Press, OxfordGoogle Scholar
Biswas AK, Tandon S, Beura CK (2016) Simple extraction method for determination of different domains of calpain and calpastatin from chicken blood and their role in post-mortem ageing of breast and thigh muscles at 4±1 °C. Food Chem 200:315–321CrossRefGoogle Scholar