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Full Description
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.
Contents
Cytochrome P450 Nomenclature, 2004.- Spectral Analyses of Cytochromes P450.- Establishment of Functional Human Cytochrome P450 Monooxygenase Systems in Escherichia coli.- Purification of Cytochromes P450.- Expression of Recombinant Flavin-Containing Monooxygenases in a Baculovirus/Insect Cell System.- Cytochrome P450 Reconstitution Systems.- Catalytic Assays for Human Cytochrome P450.- Enzymatic Analysis of cDNA-Expressed Human CYP1A1, CYP1A2, and CYP1B1 With 7-Ethoxyresorufin as Substrate.- Spectrofluorometric Analysis of CYP2A6-Catalyzed Coumarin 7-Hydroxylation.- Determination of CYP2B6 Component of 7-Ethoxy-4-Trifluoromethylcoumarin O-Deethylation Activity in Human Liver Microsomes.- High-Performance Liquid Chromatography Analysis of CYP2C8-Catalyzed Paclitaxel 6?-Hydroxylation.- Determination of CYP2C9-Catalyzed Diclofenac 4?-Hydroxylation by High-Performance Liquid Chromatography.- CYP2C19-Mediated (S)-Mephenytoin 4?-Hydroxylation Assayed by High-Performance Liquid Chromatography With Radiometric Detection.- CYP2D6-Dependent Bufuralol 1?-Hydroxylation Assayed by Reverse-Phase Ion-Pair High-Performance Liquid Chromatography With Fluorescence Detection.- Spectrophotometric Analysis of Human CYP2E1-Catalyzed p-Nitrophenol Hydroxylation.- Thin-Layer Chromatography Analysis of Human CYP3A-Catalyzed Testosterone 6?-Hydroxylation.- Determination of CYP4A11-Catalyzed Lauric Acid 12-Hydroxylation by High-Performance Liquid Chromatography With Radiometric Detection.- An Isocratic High-Performance Liquid Chromatography Assay for CYP7A1-Catalyzed Cholesterol 7?-Hydroxylation.- Use of 7-Ethoxycoumarin to Monitor Multiple Enzymes in the Human CYP1, CYP2, and CYP3 Families.- Benzydamine N-Oxygenation as a Measure of Flavin-Containing Monooxygenase Activity.- ProchiralSulfoxidation as a Probe for Flavin-Containing Monooxygenases.- Targeting Antipeptide Antibodies Toward Cytochrome P450 Enzymes.- The Human Cytochrome P450 Allele Nomenclature Committee Web Site.- Genotyping for Cytochrome P450 Polymorphisms.- Hepatocyte Cultures in Drug Metabolism and Toxicological Research and Testing.- Isolation of Rat Hepatocytes.- Rat Hepatocyte Cultures.- Rat Hepatocyte Cultures.- Hepatocytes in Suspension.- Isolation of Rat Bone Marrow Stem Cells.- Transfection of Primary Cultures of Rat Hepatocytes.- Human Hepatocyte Culture.- Determination of Cellular Localization of Expression of Flavin-Containing Monooxygenase Genes in Mouse Tissues by In Situ Hybridization.- Deletion of Genes From the Mouse Genome Using Cre/loxP Technology.- Characterization of Targeted Mouse Embryonic Stem Cell Chromosomes.- Microinjection of Targeted Embryonic Stem Cells and Establishment of Knockout Mouse Lines for Fmo Genes.- Use of Reporter Genes to Measure Xenobiotic-Mediated Activation of CYP Gene Transcription.
