Calpain: Methods and Protocols

Description

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 more about this developing field, and get inspired to make new discoveries that will aid in diagnosing and treating calpain-related diseases. Front Matter ....Pages i-xii Front Matter ....Pages 1-1 Single-Step Purification of Calpain-1, Calpain-2, and Calpastatin Using Anion-Exchange Chromatography (Ashim Kumar Biswas, Samarth Tandon)....Pages 3-11 Bacterial Expression and Purification of Calpains (Christian-Scott E. McCartney, Peter L. Davies)....Pages 13-27 Front Matter ....Pages 29-29 Casein Zymography for Analysis of Calpain-1 and Calpain-2 Activity (Ashim Kumar Biswas, Samarth Tandon)....Pages 31-38 FRET-Based Assays to Determine Calpain Activity (Christian-Scott E. McCartney, Peter L. Davies)....Pages 39-55 In Vivo Monitoring of Calpain Activity by Forster Resonance Energy Transfer (William Lostal, Daniel Stockholm, Isabelle Richard)....Pages 57-66 Determining Temporal and Spatial Expression of Calpains in Amphibians (Anna Charalambous, Ioanna Antoniades, Neophytos Christodoulou, Sara Zanardelli, Paris A. Skourides)....Pages 67-79 Immunohistochemical Localization of Calpains in the Amphibian Xenopus laevis (Jean-Marie Exbrayat, Elara N. Moudilou, Claire Brun)....Pages 81-92 Expression and Activity of Calpain A in Drosophila melanogaster (Maira Cardoso, Danielle Oliveira, Helena Araujo)....Pages 93-101 Use of the β-Glucuronidase (GUS) Reporter System to Localize Promoter Activities of the Endogenous Plant Calpain DEFECTIVE KERNEL1 (DEK1) (Zhe Liang, Hilde-Gunn Opsahl-Sorteberg)....Pages 103-108 Front Matter ....Pages 109-109 LabCaS for Ranking Potential Calpain Substrate Cleavage Sites from Amino Acid Sequence (Yong-Xian Fan, Xiaoyong Pan, Yang Zhang, Hong-Bin Shen)....Pages 111-120 CalCleaveMKL: a Tool for Calpain Cleavage Prediction (David A. duVerle, Hiroshi Mamitsuka)....Pages 121-147 Identification of Calpain-Activated Protein Functions (Maria del Carmen Lafita-Navarro, Maralice Conacci-Sorrell)....Pages 149-160 Front Matter ....Pages 161-161 Production and Purification of Recombinant Calpastatin (Roberta De Tullio, Monica Averna)....Pages 163-185 Isolation of Endogenous Calpastatin (Roberta De Tullio, Monica Averna)....Pages 187-194 Immunoblotting for Calpastatin Expression (Monica Averna, Roberta De Tullio)....Pages 195-206 Front Matter ....Pages 207-207 Experimental Manipulation of Calpain Activity In Vitro (Seiichi Kitagawa)....Pages 209-218 In Vivo Calpain Knockdown Using Delivery of siRNA (Xiaoqian Liu, Guochang Hu)....Pages 219-232 In Vivo Expression of Mutant Calpains in the Eye Using Lentivirus (Katherine J. Wert, Vinit B. Mahajan)....Pages 233-247 Methods of Calpain Inhibition to Determine the Role of Calpains in Embryo Development in Amphibians (Ioanna Antoniades, Anna Charalambous, Neophytos Christodoulou, Sara Zanardelli, Paris A. Skourides)....Pages 249-259 Genetic Models of Calpain Deficiency and Ectopic Expression (Yan Gao, Christine Hall, James MacLeod, Peter A. Greer)....Pages 261-274 Back Matter ....Pages 275-276