An Introduction of Protein.ppt

An Introduction to Protein/Peptide Microarray Zhonghua Liu 01/03/2011 DNA mRNA Protein Proteins 20 amino acids 30,000 – 34,000 genes 2,000,000 proteins Protein Functions Signal transduction Transcription regulation Immune response Other vital cellular actions Proteomics An organism’s proteome: a catalog of all proteins expressed throughout life expressed under all conditions The goals of proteomics: to catalog all proteins to understand their functions to understand how they interact with each other Methods for Protein Analysis Gel electrophoresis, northern/western blot (fluorescence/radio active label) X-ray crystallography 结晶学 2D - mass spectrometry光谱法 Protein microarrays基因芯片 1. High throughput analysis of hundreds of thousands of proteins. 2. Proteins are immobilized固定 on glass chip. 3. Various probes (protein, lipids, DNA, peptides, etc) are used. Protein Array VS DNA Microarray Protein Array Fabrication制造 Protein Interactions Probes (antibody) on surface recognize target proteins. Identification of expressed proteins from samples. Typical quantification method for large # of expressed proteins. Technical Challenges in Protein/Peptide Chips Protein Attachment PepArray– a user-driven PepArray design program Method: Protein Detection through Domain Binding Recombinant SH2 protein and cell lysate binding assays Dye-conjugated secondary Ab Peptide on chip Dye-labeled antibody P P P His-SH2 His-SH2 Recombinant protein binding assay Primary antibody P P P Cell lysate SH2 protein or complex Peptide on chip Cell lysate binding assay or dye-conjugated secondary Ab SH2 Proteins and Cell Lines Studied in This Work This indicates that Grb2 could bind specifically to the peptides synthesized on chip. SH2 Protein Binds to SH2-PPEP Microarray Recombinant SH2 Protein Binding By using four recombinant SH2 proteins, specific and distinct binding profiles were obtained. The consensus sequences are consistent with those reported in literatures. Rec