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For this second edition of their much praised Cytochrome P450, the editors have collected accounts of the essential core techniques that use the latest methodologies for the investigation of P450s. Highlights include protocols for spectral analysis and purification of P450s, enzymatic assays of P450s and flavin-containing monooxygenases (FMOs), expression of P450s and FMOs in heterologous systems, and the production and use of antipeptide antibodies. Additional chapters contain readily reproducible techniques for the transfection of hepatocytes for gene regulation studies, P450 reporter gene assays, in situ hybridization, and analysis of genetic polymorphisms. Although the emphasis is on P450s of mammalian origin, many of the readily reproducible methods described are suitable for P450s from any source.
The Role of Methods in the Discovery of the Cytochromes P450 Ronald W. Estabrook . Cytochrome P450 Nomenclature David R. Nelson . Spectral Analyses of Cytochromes P450 John B. Schenkman and Ingela Jansson . Purification of Cytochromes P450: Rat and Human Hepatic Forms F. Peter Guengerich and Martha V. Martin. Isolation and Purification of Constitutive Forms of Microsomal Cytochrome P450 John B. Schenkman and Ingela Jansson .Purification of Extrahepatic Cytochromes P450 Margaret Warner and Jan-?ke Gustafsson. Purification of Cytochromes P450: Products of Bacterial Recombinant Expression Systems F. Peter Guengerich Natalie A. Hosea and Martha V. Martin.yCytochrome P450 Reconstitution SystemsTsutomu Shimada and Hiroshi Yamazaki . Catalytic Assays for HumanyCytochromeyP450 Thomas K.H. Chang andyDavidyJ.yWaxman.yEnzymaticyAnalysisyofycDNA- ExpressedyHumanyCYP1A1yCYP1A2yandyCYP1B1ywith 7-Ethoxyresorufin as SubstrateThomas K. H. Chang and David J. Waxman. Spectrofluorometric Analysis of CYP2A6-Catalyzed Coumarin 7- Hydroxylation DavidyJ.Waxman and Thomas K. H. Chang. Determination of the CYP2B6 Component of 7-Ethoxy-4-Trifluoromethylcoumarin O-Deethylation Activity in Human Liver Thomas K. H. Chang Charles L. Crespi and David J. Waxman. High-Performance Liquid Chromatographic Analysis of YP2C8-Catalyzed Paclitaxel 6a-HydroxylationCharles L. Crespi Thomas K. H. Chang and David J. Waxman. Determination of CYP2C9-Catalyzed Diclofenac 4'- Hydroxylation by High-Performance Liquid ChromatographyCharles L. Crespi Thomas K. H. Chang and David J. Waxman. CYP2C19-Mediated (S)-Mephenytoin 4'-Hydroxylation Assayed by High- Performance Liquid Chromatography with Radiometric DetectionCharles L. Crespi Thomas K. H. Chang and David J. Waxman. CYP2D6-Dependent Bufuralol 1'-Hydroxylation Assayed by Reversed- Phase Ion-Pair High-Performance Liquid Chromatography with Fluorescence DetectionCharles L. Crespi Thomas K. H. Chang andyDavid J.yWaxman. Spectrophotometric Analysis of Human YP2E1- Catalyzed p-Nitrophenol HydroxylationThomas K. H. Chang Charles L. Crespi and David J. Waxman. Thin-Layer Chromatographic Analysis of Human CYP3A-Catalyzed Testosterone 6b- Hydroxylation David J. Waxman and Thomas K. H. Chang. Determination of CYP4A11-Catalyzed Lauric Acid 12-Hydroxylation by High-Performance Liquid Chromatography with Radiometric Detection Charles L. Crespi Thomas K. H. Chang and David J. Waxman. An Isocratic High- Performance Liquid Chromatographic Assay for CYP7A1-Catalyzed Cholesterol 7a- HydroxylationDavid J. Waxman and Thomas K. H. Chang. Use of 7-Ethoxycoumarin to Monitor Multiple Enzymes in the Human CYP1 CYP2 and CYP3 Families David J. Waxman and Thomas K. H. Chang. Expression of Eukaryotic Cytochromes P450 in E. Coli.Christopher M. Jenkins Irina Pikuleva and Michael R. Waterman. Expression of Mammalian Cytochromes P450 in YeastYoshiyasu Yabusaki. Expression of Cytochromes P450 in a Baculovirus System Steven R. Hood Girish Shah and Peter Jones. Expression of Cytochromes P450 in Mammalian Cells Els M. de Groene. Monoclonal Antibodies to Cytochromes P450 Harry V. Gelboin Magang Shou Inna Goldfarb Tian J. Yang and Kristopher Krausz. Targeting Antipeptide Antibodies Towards Cytochrome P450 Enzymes Robert J. Edwards. Signals for Retention of Cytochromes P450 in the Endoplasmic Reticulum Elzbieta Skorupa and Byron Kemper. Quantification of Cytochrome P450 Gene Expression by RNase Protection AnalysisColin N. Palmer. Hepatocyte Cultures in Drug Metabolism and Toxicological Research and TestingVera Rogiers and Antoine Vercruysse. Isolation of Rat Hepatocytes Karen De Smet Sonja Beken Tamara VanHaecke Marleen Pauwels Antoine Vercruysse and Vera Rogiers. Collagen Gel Cultures of Rat Hepatocytes: Collagen Gel Sandwich and Immobilization Cultures Sonja Beken Tamara VanHaecke Karen De Smet Marleen Pauwels Antoine Vercruysse and Vera Rogiers. Rat Hepatocyte Cultures: Conventional Monolayer Cultures and Cocultures with Rat Liver Epithelial CellsTamara VanHaecke Karen De Smet Sonja Beken Marleen Pauwels Antoine Vercruysse and Vera Rogiers. Isolation of Periportal and Pericentral Hepatocytes Rolf Gebhardt. Perifusion Culture of Hepatocytes Rolf Gebhardt. Human Hepatocyte Culture Jean Bernard Ferrini Jean-Claude Ourlin Lydiane Pichard G'rard Fabre and Patrick Maurel . Reconstruction of Human Epidermis In Vitro Marie-C'cile Lenoir-Viale. Gene Transfer and Expression Frank Gaunitz and Monika Papke. Lipid-Mediated Gene Transfer into Normal Adult Human Hepatocytes in Primary Culture Jean-Claude Ourlin Marie-Jos' Vilarem Martine Daujat Marie-C'cile Harricane and Patrick Maurel. Analysis of DNA-Protein Interactions by Gel RetardationHelen Dell Siew Cheng Wong Ian R. Phillips and Elizabeth A. Shephard . Cytochrome P450 Gene Regulation: Analysis of Protein-DNA Interactions In Situ Steve T. Okino and James P. Whitlock Jr. .Analysis of Cytochrome P450 Polymorphisms Ann K. Daly Sophia C. Monkman Joanne Smart Annette Steward and Suzanne Cholerton. Induction of Cytochrome P4501A (CYP1A) in Fish: A Biomarker for Environmental Pollution Bente M. Nilsen Karin Berg and Anders Goks>yr. Generation of Allelic Series in Mice by Gene Targeting Maureen Bunger and Christopher Bradfield. Index.
From Reviews of the First Edition... "...a remarkable book, highly useful for all our scientific readers..." -Cellular and Molecular Biology "...will be welcomed by laboratory scientists of various profiles who need authoritative protocols for their studies." -Modern Pathology From the reviews of the second edition: "In this second edition, editors Phillips and Shephard have updated the text to reflect current advances and to include chapters on some more contemporary procedures ! . I'm sure that the wealth of background information in the book would be valuable. Overall ! this is a useful reference book for experienced researchers and for those new to this discipline. Recommended." (David Wilkinson, British Toxicology Society Newsletter, Winter, 2006)