Calpain Methods and Protocols

The purpose of Calpain Methods and Protocols is quite straightf- ward: it is to present the actual experimental methods used in many different laboratories for the study of calpain. It will provide the vital experimental detail, and the discussion of possible pitfalls, for which the standard journals no longer provide space. This will make it as easy as possible for investi- tors interested in calpain to adopt established methods without repeating old mistakes, and to adapt and apply these methods in novel approaches to the many outstanding calpain questions. These questions range from purely biochemical problems of protein structure and enzyme regulation at the molecular level, through large areas of cell biology, to applied and clinical aspects of calpain function in human d- ease. Within this panoply of topics, a wide range of investigators will find many fascinating and as yet unanswered questions about calpain. Calpain Methods and Protocols will provide instant access to many essential te- niques, while saving them the time and effort involved in developing a new method. In addition to questions relating to the normal physiological roles of the calpains, there is considerable evidence that inappropriate calpain activity may have pathological effects in many tissues, for example, following ischemia. This provides a major stimulus for the development of specific calpain inhi- tors for therapeutic purposes, and for the development of methods to evaluate such inhibitors.

Includes supplementary material: sn.pub/extras

Klappentext

John S. Elce and a seasoned team of principal investigators present a set of proven and easily followed protocols for studying calpain. The methods include in vitro techniques for the detection, expression, purification, and assay of µ- and m-calpain, supplemented with a wide range of system and tissue models for studying both the physiological functions of, and the effects of inhibitors on, calpain. The systems used include neural tissue, kidney, liver, the eye, and membrane fusion in muscle and erythrocytes, each in connection with hypoxia or other injury. Among the analytical techniques employed are casein zymography, immunofluorescence, and calpain activity assays. Highly practical and readily reproducible, Calpain Methods and Protocols offers investigators involved in basic and clinically oriented calpain research a gold-standard collection of powerful experimental tools for discovering the nature and functions of calpains.

Zusammenfassung
"This book collects contributions from essentially all leading specialists in the area and the chapters are constructed in a way that should make it a useful bench-top companion. The book is good value for money: 36 chapters for $89 should make it an all but mandatory acquisition for libraries, and especially laboratories having programs on cell biology and cell pathology." -Cell Biology International

Inhalt
Purification of Components of the Calpain System.- Purification of µ- and m-Calpain and Calpastatin from Animal Tissues.- A Simple Protocol for Separation and Assay of µ-Calpain, m-Calpain and Calpastatin from Small Tissue Samples.- Purification and Quantification of Calcium-Activated Neutral Proteases I and II and Novel Isoforms from Cultured Osteoblastic Cells by Ion-Exchange Fast Protein Liquid Chromatography.- Purification of Calpain by Affinity Chromatography on Reactive Red-Agarose or on Casein-Sepharose.- Affinity Purification of ?-Calpain from Erythrocytes on an Immobilized Peptide from the Plasma Membrane Calcium Pump.- Expression of m-Calpain in Escherichia coli.- Purification and Characterization of Crustacean Calpain-like Proteinases.- Drosophila Cal pains.- Molecular Analysis of p94 and Its Application to Diagnosis of Limb Girdle Muscular Dystrophy Type 2A.- Purification of Recombinant Calpastatin Expressed in Escherichia coli.- Preparation of Calpastatin Samples for Western Blotting.- Isolation and Characterization of Calpain Activator Protein from Bovine Brain.- Assays of Calpain.- Calpain Zymography with Casein or Fluorescein Isothiocyanate Casein.- Casein Zymogram Assessment of ?-Calpain and m-Calpain Activity After Traumatic Brain Injury in the Rat In Vivo.- Fluorescence Measurements of Ca2+ Binding to Domain VI of Calpain.- Kinetic Analysis of Human ?-Calpain Autolysis.- A Sensitive and Continuous Fluorometric Activity Assay Using a Natural Substrate.- Measurement of Calpain Activity In Vitro and In Situ Using a Fluorescent Compound andTau as Substrates.- Localization of Calpain by Immunofluorescence in Adherent Cells.- A Radioimmunologic Technique for Assessing Calpain Activation in Cells.- Specific Tissues.- Calpains and Myogenesis.-Calpastatin (the Endogenous Calpain Inhibitor) and Membrane Protein Degradation in Cell Fusion.- The Role of Calpain in Neurofilament Protein Degradation Associated With Spinal Cord Injury.- Calpain-Mediated Truncation of Glutamate Ionotropic Receptors.- Concurrent Assessment of Calpain and Caspase-3 Activity by Means of Western Blots of Protease-Specific Spectrin Breakdown Products.- Rat Renal Proximal Tubules, Hypoxia, lonomycin, and Calpain.- Calpain Activity in Rat Renal Proximal Tubules.- Calpain Activity in Rat Renal Proximal Tubules.- Cellular In Vivo Assay of Calpain Activity Using a Fluorescent Substrate.- Calpain Methods in Hepatic Ischemia-Reperfusion Injury.- Myocardial Ischemia-Reperfusion Injury and Proteolysis of Fodrin, Ankyrin, and Calpastatin.- Calpains in the Lens and Cataractogenesis.- Specific Substrates.- Proteolysis of Cortactin by Calpain in Platelets and In Vitro.- Proteolysis of p53 Protein by Ubiquitous Calpains.- Modulation of Calpain-Mediated Protein Kinase C Activation Within Intact Cells.- Strategies for Regulating Calpain Activities in Living Cells.- Assays of Apoptosis.