分子生物图谱.ppt

antisense Gene Transcription 1 Overview of Gene Expression 2 Overview of Transcription 3 Gene’s Regulatory Elements 4 Transcription Mechanism in Prokaryotes 5 Transcription Mechanism in Eukaryotes 6 Motif Structure of Transcription Factors 7 Histone Acetylation and DNA methylation 8 Regulation of Transcription Factors 9 Transcription of RNA Genes 10 Genome Replication of Viruses + Charge 2: Electrophoresis Protein migrate at different position depending on their net charge Protein structure Charge 3: Isoelectric focusing A protein will stop moving at position corresponding to its isoelectric point (pI) in a pH gradient gel. Protein structure 3. Hydrophobicity: hydrophobic interaction chromatography Similar to ion-exchange chromatography except that column material contains aromatic or aliphatic alkyl groups Protein structure 4. Affinity chromatography Enzyme-substrate binding Receptor-ligand binding Antibody-antigen binding Protein structure 5. Recombinant techniques: Clone the interested protein encoding gene in an expression vector with a purification tag added at the 5’- or 3’ end of the gene Protein overexpression in a cell Protein purification with affinity chromatography. Protein structure Mass Determination ?Gel filtration chromatography and SDSComparing of the unknown protein with a proper standard Popular SDS: cheap and easy with a 5-10% error SDS: sodium dodecyl sulfate, makes the proteins negatively charged and the overall charge of a protein is dependent on its mass. Protein structure Mass spectrometry: Molecules are vaporized and ionized, and the degree of deflection (mass-dependent) of the ions in an electromagnetic field is measured extremely accurate, but expensive MALDI can measure the mass of proteins smaller than 100 KDa Helpful to detect post-translational modification Protein sequencing: relying on the protein data base Protein sequencing : Determine the primary structure of a protein Specific enzyme/chemical cleavage: Trypsin