Full Description
Since the discovery of p53 as a tumor suppressor, numerous methods have evolved to reveal the unique structural features and biochemical functions of this protein. Several unique properties of p53 posed a challenge to understa- ing its normal function in the initial phase of its research. The low levels of p53 in normal cells, its stabilization under situations of genotoxic stress, induction of growth arrest, and apoptosis with stabilization of the protein, obstructed the visibility of its normal, unmutated function. The property of p53 that can sense a promoter and transactivate or inhibit is still not well understood. It is still not known whether it is the absence of the protein that causes tumorigenesis, or if its mutants have a dominant role in inducing cancer. p53 Protocols comprises eighteen chapters for the study of the diverse properties of p53 and related proteins. The methods included are invaluable for delineating the function of other proteins that may function as tumor suppr- sors or growth suppressors. The chapters are not presented in any schematic order, for the importance and diversity of the functions of p53 make it imp- sible to organize them suitably. We have made a sincere effort to collect the methods most useful to those investigators working on tumor suppressors or growth suppressors. The purpose of p53 Protocols is not only to provide investigators with methods to analyze similar biochemical functions, but also to familiarize them with the associated problems that arose during the course of investigations.
Contents
Adenovirus Expressing p53.- Purification of Recombinant p53 from Sf9 Insect Cells.- Generation and Characterization of p53 Mutant Mice.- Identification of p53 Target Genes by Fluorescent Differential Display.- p53-Induced Gene Expression Analysis.- Real-Time Polymerase Chain Reaction Quantitation of Relative Expression of Genes Modulated by p53 Using SYBR® Green I.- Transactivation and Transrepression Studies with p53.- Methods to Study p53-Repressed Promoters.- Interaction of p53 with Cellular Proteins.- Interaction of p53 with the Adenovirus E1B-55 kDa Protein.- In Vivo Footprinting and DNA Affinity Chromatography for Analysis of p53 DNA Binding Ability.- Signaling to p53.- Detection of Mitochondrial Localization of p53.- Analysis of Nuclear and Cytoplasmic Degradation of p53 in Cells after Stress.- Mutational Analysis of p53 in Human Tumors.- Mutational Analysis of p53 in Human Tumors.- Flow Cytometric Analysis of p53-Induced Apoptosis.- Flow Cytometric Analysis of MDM2-Mediated Growth Arrest.
