国家人类基因组北方研库究中心.pptVIP

Protein Complex and Protein-protein Interaction 彭鲲鹏 国家人类基因组北方研究中心 Email: pengkp@ Central dogma: the story of life Proteins function in association with other proteins or biomolecules, but not in isolation Introduction to Proteomics the analysis of genomic complements of proteins dynamic systematic discovery-driven Goals of Proteomics Types of Proteomics Expression Proteomics Quantitative study of protein expression and their changes between samples that differs by some variable Functional Proteomics To study protein-protein interaction, 3-D structures, cellular localization and PTMs in order to understand the physiological function of the whole set of proteome. Approaches Blue Native PAGE separation of native proteins in complex. Coomassie Blue G: stable and negatively charge multiprotein complex. 6-aminocaproic acid: solubilize membrane protein complex instead of salts. the resolution is not so high that the prepurification is needed. Blue Native PAGE Blue Native PAGE Blue Native PAGE of chloroplast thylakoid membranes Blue Native PAGE of chloroplast thylakoid membranes Blue Native PAGE of multiprotein complex from whole cellular lysate Identification and analysis of distinct proteasomes by WCL 2D BN/SDSVisualization of MPCs on a 2D WCL BN/SDS gel Far Western Max: functional cloning of a Myc-binding protein Association of Rb with HIP1 GST Pulldown Interactions of Cellular Polypeptides with the Cytoplasmic Domain of the Mouse Fas Antigen GST-mFas fusion proteins GST-mFas-associated polypeptides from 32S-labeled HeLa, L929, and Jurkat cell lysates GST-mFas-associated polypeptides are stable to high salt concentrations Association is blocked by preincubation with a polyclonal antibody against GST-mFas Differential association with mutant forms of GST-mFas Schematic representation of the mouse Fas antigen and its binding proteins Epitope tagging Co-Immunoprecipitation Co-Immunoprecipitation False positive and control Reduction of nonspecific protein background Bindi