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Calpain Methods and Protocols

  • Kartonierter Einband
  • 342 Seiten
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The purpose of Calpain Methods and Protocols is quite straightf- ward: it is to present the actual experimental methods used in ma... Weiterlesen
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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.


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.


Part I. Purification of Components of the Calpain System. Purification of µ- and m-Calpain and Calpastatin from Animal Tissues, Valery F. Thompson and Darrel E. Goll. A Simple Protocol for Separation and Assay of µ-Calpain, m-Calpain and Calpastatin from Small Tissue Samples, Jan-Olof Karlsson. 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, Elsa J. B. Murray, Keyvan Behnam, Mario S. Grisanti, and Samuel S. Murray. Purification of Calpain by Affinity Chromatography on Reactive Red-Agarose or on Casein-Sepharose, Dorothy E. Croall. Affinity Purification of µ-Calpain from Erythrocytes on an Immobilized Peptide from the Plasma Membrane Calcium Pump: Some Studies on Erythrocyte µ-Calpain, Maurizio Molinari and Ernesto Carafoli. Bacterial Expression and Purification of Recombinant m-Calpain, John S. Elce. Purification and Characterization of Crustacean Calpain-Like Proteinases, Donald L. Mykles. Drosophila Calpains: Purification of a Calpain-Like Enzyme from Fruit Flies, and Expression in E. coli, Gáspár Jékely, Marianna Pintér, and Peter Friedrich. Molecular Analysis of p94 and Its Application to Diagnosis of Limb Girdle Muscular Dystrophy Type 2A, Hiroyuki Sorimachi, Yasuko Ono, and Koichi Suzuki. Purification of Recombinant Calpastatin Expressed in Escherichia coli, Masatoshi Maki and Kiyotaka Hitomi. Preparation of Calpastatin Samples for Western Blotting, Masatoshi Maki and Kiyotaka Hitomi. Isolation and Characterization of Calpain Activator Protein from Bovine Brain, Edon Melloni, Mauro Michetti, Franca Salamino, Roberto Minafra, Bianca Sparatore, and Sandro Pontremoli. Part II. Assays of Calpain. Calpain Zymography with Casein or FITC-Casein, J. Simon C. Arthur and Donald L. Mykles. Casein Zymograph Assessment of µ- and m-Calpain Activity After Traumatic Brain Injury in the Rat In Vivo, Xiurong Zhao, Jennifer K. Newcomb, Brian R. Pike, and Ronald L. Hayes. Fluorescence Measurement of Ca2+ Binding to Domain VI of Calpain, J. Simon C. Arthur and John S. Elce. Kinetic Analysis of Human µ-Calpain Autolysis, Peter Tompa and Peter Friedrich. A Sensitive and Continuous Fluorometric Activity Assay Using a Natural Substrate, MAP2, Peter Tompa, Éva Schád, and Peter Friedrich. Measurement of Calpain Activity In Vitro and In Situ Using a Fluorescent Compound and Tau as Substrates, Rodney P. Guttmann and Gail V.W. Johnson. Localization of Calpain by Immunofluorescence in Adherent Cells, Sucheta Kulkarni and Joan E. B. Fox. A Radioimmunologic Technique for Assessing Calpain Activation in Cells, Ronald L. Mellgren. Part III. Specific Tissues. Calpains and Myogenesis, Patrick Cottin, Sylvie Poussard, Elise Dargelos, Denis Balcerzak, Bernadette Aragon, Jean Jacques Brutis, and André Ducastaing. Calpastatin (the Endogenous Calpain Inhibitor) and Membrane Protein Degradation in Cell Fusion, Nechama S. Kosower and Sivia Barnoy. The Role of Calpain in Neurofilament Protein Degradation Associated with Spinal Cord Injury, Naren L. Banik and Donald C. Shields. Calpain-Mediated Truncation of Glutamate Ionotropic Receptors: Methods for Studying the Effects of Calpain Activation in Brain Tissue, Xiaoning Bi, Ruifen Bi, and Michel Baudry. Concurrent Assessment of Calpain and Caspase-3 Activity by Means of Western Blots of Protease-Specific Spectrin Breakdown Products, Jennifer K. Newcomb, Brian R. Pike, Xiurong Zhao, and Ronald L. Hayes. Rat Renal Proximal Tubules, Hypoxia, and Calpain, Charles L. Edelstein. Calpain Activity in Rat Renal Proximal Tubules: An In Vitro Assay, Charles L. Edelstein. Calpain Activity in Rat Renal Proximal Tubules: An In Situ Assay, Charles L. Edelstein. Cellular In Vivo Assay of Calpain Activity Using a Fluorescent Substrate: Application to Study of Anoxic Liver Injury, Barry G. Rosser and Gregory J. Gores. Calpain Methods in Hepatic Ischemia-Reperfusion Injury, David Sindram and Pierre-Alain Clavien. Myocardial Ischemia-Reperfusion Injury and Proteolysis of Fodrin, Ankyrin, and C


Titel: Calpain Methods and Protocols
EAN: 9781617371059
ISBN: 978-1-61737-105-9
Format: Kartonierter Einband
Genre: Medizin
Anzahl Seiten: 342
Gewicht: g
Größe: H229mm x B229mm x T152mm
Jahr: 2010
Auflage: Softcover of Or



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