Author : David R. Nelson (auth.), Ian R. Phillips, Elizabeth A. Shephard (eds.)
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. 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.