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Title Cytochrome P450 protocols / edited by Ian R. Phillips, Elizabeth A. Shephard
Edition 2nd ed
Published Totowa, N.J. : Humana Press, ©2006


Description 1 online resource (xiii, 363 pages) : illustrations
Series Methods in molecular biology ; 320
Methods in molecular biology (Clifton, N.J.) ; 320
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: products of bacterial recombinant expression systems -- Expression of recombinant flavin-containing monooxygenases in a baculovirus/insect cell system -- Cytochrome P450 reconstitution systems -- Catalytic assays for human cytochrome P450: an introduction -- 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[alpha] [alph]-hydroxylation -- Determination of CYP2C9-catalyzed diclofenac 4¹-hydroxylation by high-performance liquid chromatography -- CYP2C19-mediated (S)-mephenytoin 4¹-hydroxylation assayed by high-performance liquid chromatography with radiometric detection -- CYP2D6-dependent bufuralol 1¹-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[beta][beta]-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[alpha][alpha]-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 -- Prochiral sulfoxidation as a probe for flavin-containing monooxygenases -- Targeting antipeptide antibodies toward cytochrome P450 enzymes -- The human cytochrome P450 allele nomenclature committee web site: submission criteria, procedures, and objectives -- Genotyping for cytochrome P450 polymorphisms
Hepatocyte cultures in drug metabolism and toxicological research and testing -- Isolation of rat hepatocytes -- Rat hepatocyte cultures: conventional monolayer and cocultures with rat liver epithelial cells -- Rat hepatocyte cultures: collagen gel sandwich and immobilization 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 the expression of flavin-containing monooxygenase genes in mouse tissues by In Situ hybridization -- Deletion of genes from the mouse genome using Cr/loxP technology -- Characterization of targeted mouse embryonic stem cell chromosomes: karyotyping and fluorescence in situ hybridization of metaphase spreads -- 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
Summary 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. Also described are techniques for the generation of mice with targeted gene disruptions. Although the emphasis is on P450s of mammalian origin, many of the readily reproducible methods described are suitable for P450s from any source. Each chapter is written by researchers who have been involved in the development and application of the particular technique to P450s or FMOs. The protocols are presented in a step-by-step manner, with extensive cross-references to notes that highlight critical steps, potential problems, and alternative methods so that the researcher can understand the basis of the method and perform it successfully. Cutting-edge and highly practical, Cytochrome P450, Second Edition provides both novices and experienced researchers across many fields all the tools needed today to elucidate the crucial biological role played by cytochrome P450 in the metabolism of therapeutic drugs, chemical carcinogens, and environmental pollutants
Bibliography Includes bibliographical references and index
Notes Print version record
Subject Cytochrome P-450 -- Laboratory manuals
High performance liquid chromatography.
Liver cells.
Cytochrome P-450.
Chromatography, High Pressure Liquid
Cytochrome P-450 Enzyme System
Mixed Function Oxygenases
SCIENCE -- Life Sciences -- Biochemistry.
Cytochrome P-450
Cytochroom P450.
Wetenschappelijke technieken.
Genre/Form Handbooks and manuals
Laboratory manuals.
Manuels de laboratoire.
Form Electronic book
Author Phillips, Ian R. (Ian Randall), 1951-
Shephard, Elizabeth A. (Elizabeth Anne), 1950-
ISBN 1588294412