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Assaying Calpain Activity

Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 370)

Abstract

The calpains represent a well-conserved family of intracellular proteases that exhibit broad substrate specificity and consequently influence many cellular processes. Calpain activity regulates the turnover of integrin-linked focal adhesions, which controls cell adhesion to extracellular matrix substrates, cell migration across such substrates, as well as the signalling output from the associated integrin receptors. Thus, calpain activity regulates both the physical interaction and biochemical communication between cells and the extracellular environment that is vital for normal cell function. Modulation of calpain activity is associated with a number of human diseases, and recent studies have identified calpain activity as an important therapeutic target in several disease areas. These studies have driven the development of in vitro and live-cell-based assays for high-throughput identification and evaluation of calpain inhibitors. However, many of the unique features of calpain activity devalue the physiological relevance of existing assays. In this chapter we describe a modified approach that monitors calpain activity by taking into account several of the factors that control calpain activity and substrate specificity under physiological conditions. The development of such second-generation assays may help to identify more effective calpain intervention strategies.

Key Words

Calpain Src focal adhesion kinase talin cleavage sodium dodecyl sulfate-polyacrylamide gel electrophoresis phosphorylation fluorescence resonance energy transfer biosensor 
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Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  1. 1.Cell Adhesion-Linked Kinase LaboratoryBeatson Institute for Cancer ResearchBearsden, GlasgowUK

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