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This volume brings together a plethora of protocols and experimental methods used by scientists to study calpains, their inhibitors, and their substrates. It also explores bioinformatic approaches to calpain substrate identification. The chapters in this book are divided into five parts and cover topics such as production and purification of calpains; determination of calpain localization, expression, and activity; identification of calpain-activated protein function; interrogation of calpastatin; and manipulation of calpain expression. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Cutting-edge and practical, Calpain: Methods and Protocols is a valuable resource for researchers and scientists who want to learn moreabout this developing field, and get inspired to make new discoveries that will aid in diagnosing and treating calpain-related diseases.

Calpain

Single-Step Purification of Calpain-1, Calpain-2, and Calpastatin using Anion Exchange Chromatography.- Bacterial Expression and Purification of Calpains.- Casein Zymography for Analysis of Calpain-1 and Calpain-2 Activity.- FRET-Based Assays to Determine Calpain Activity.- In Vivo Monitoring of Calpain Activity by Forster Resonance Energy Transfer.- Determining Temporal and Spatial Expression of Calpains in Amphibians.- Immunohistochemical Localization of Calpains in the Amphibian Xenopus laevis.- Expression and Activity of Calpain A in Drosophila melanogaster.- Use of the beta-Glucoronidase Reporter System (GUS) to Localize Promoter Activities of the Endogenous Plant Calpain DEFECTIVE KERNEL1 (DEK1).- LabCaS for Ranking Potential Calpain Substrate Cleavage Sites from Amino Acid Sequence.- CalCleaveMKL: A Tool for Calpain Cleavage Prediction.- Identification of Calpain-Activated protein Functions.- Production and Purification of Recombinant Calpastatin.- Isolation of Endogenous Calpastatin.- Immunoblotting for Calpastatin Expression.- Experimental Manipulation of Calpain Activity In Vitro.- In Vivo Calpain Knockdown using Delivery of siRNA.- In Vivo Expression of Mutant Calpains in the Eye using Lentivirus.- Methods of Calpain Inhibition to Determine the Role of Calpains in Embryo Development in Amphibians.- Genetic Models of Calpain Deficiency and Ectopic Expression.