Abstract
Casein zymography has become one of the gold standard assays for monitoring mammalian calcium-activated proteases (calpains) in purified enzyme, cell, or tissue samples. This calpain zymography method takes advantages of (1) casein is an excellent substrate for major isoforms of calpains (Calpain-1, 2 and 3), (2) the embedded casein is digested into small peptides where the calpain bands are located, thus creating a clear zone upon Commassie blue gel staining, and (3) the calpain isoforms have different gel mobility under native gel conditions. Casein zymography is also useful in studying reversibility of inhibitor binding to calpains.
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References
Romanic AM, White RF, Arleth AJ et al (1998) Matrix metalloproteinase expression increases after cerebral focal ischemia in rats: inhibition of matrix metalloproteinase-9 reduces infarct size. Stroke 29:1020–1030
Wang KKW (2002) Assaying proteases in cellular environments. Curr Protoc Protein Sci Chapter 21:Unit 21.12. doi:10.1002/0471140864.ps2112s27
Wang X, Mori T, Jung JC et al (2002) Secretion of matrix metalloproteinase-2 and -9 after mechanical trauma injury in rat cortical cultures and involvement of MAP kinase. J Neurotrauma 19:615–625. doi:10.1089/089771502753754082
Raser KJ, Posner A, Wang KKW (1995) Casein zymography: a method to study mu-calpain, m-calpain, and their inhibitory agents. Arch Biochem Biophys 319:211–216
Zhao X, Newcomb JK, Posmantur RM et al (1998) pH dependency of mu-calpain and m-calpain activity assayed by casein zymography following traumatic brain injury in the rat. Neurosci Lett 247:53–57
Huang Y, Wang KKW (2001) The calpain family and human disease. Trends Mol Med 7:355–362
Sorimachi H, Hata S, Ono Y (2011) Calpain chronicle—an enzyme family under multidisciplinary characterization. Proc Jpn Acad Ser B Phys Biol Sci 87:287–327
Ma H, Shih M, Hata I et al (1998) Protein for Lp82 calpain is expressed and enzymatically active in young rat lens. Exp Eye Res 67:221–229. doi:10.1006/exer.1998.0515
Azuma M, Fukiage C, Higashine M et al (2000) Identification and characterization of a retina-specific calpain (Rt88) from rat. Curr Eye Res 21:710–720
Zhao X, Posmantur R, Kampfl A et al (1998) Subcellular localization and duration of μ-calpain and m-calpain activity after traumatic brain injury in the rat: a casein zymography study. J Cereb Blood Flow Metab 18:161–167. doi:10.1097/00004647-199802000-00006
Giguere CJ, Covington MD, Schnellmann RG (2008) Mitochondrial calpain 10 activity and expression in the kidney of multiple species. Biochem Biophys Res Commun 366:258–262. doi:10.1016/j.bbrc.2007.11.133
Acknowledgment
This study is supported in part by NIH grant R21 NS085455-01 (K.K.W.).
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Wang, K.K.W. (2017). Calpain Zymography: General Methodology and Protocol. In: Wilkesman, J., Kurz, L. (eds) Zymography. Methods in Molecular Biology, vol 1626. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7111-4_26
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DOI: https://doi.org/10.1007/978-1-4939-7111-4_26
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-7111-4
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