武汉大学分子考研本科生教学ppt2008chapter21.ppt

* Three principle methods 1. 2-D gel electrophoresis for protein separation (蛋白质分离). 2. MS spectrometry for the precise determination of the molecular weight and identify of a protein (蛋白质鉴定). 3. Bioinformatics for assigning proteins and peptides to the predicted products of protein coding sequence in the genome (蛋白质确定). * Fig 20-24 * Topic 3: Study the interaction between protein and nucleic acid CHAPTER20: Techniques of Molecular Biology Gel retardation assay Nuclease protection assay * Gel retardation (凝胶阻滞) A short labeled nucleic acid is mixed with a cell or nuclear extract expected to contain the binding protein. Then, samples of labeled nucleic acid, with and without being incubated with the extract, are run on a gel. The DNA-protein complexes are shown by the presence of slowly migrating bands. DNA bound to two proteins DNA-protein complex Bare DNA A DNA bound with more than one protein to form a larger complex. * DNase I footprinting (DNase I 足迹法) Identify the actual region of sequence with which the protein interacts. 5’ * Sequence ladder is required to determine the precise position AATAAG * Bind protein DNase(mild),then remove protein and denature DNA DNase footprinting （1）The protein protects DNA from attack by DNase. （2）Treat the DNA-protein complex with DNase I under mild conditions, so that an average of only one cut occur per DNA molecule. Electrophoresis, autoradiograph * 0 1 5 [Protein] The three lanes represent DNA that was bound to 0, 1, and 5 units of protein. The lane with no protein shows a regular ladder of fragments. The lane with one unit shows some protection, and the lane with 5 units shows complete protection in the middle.By including sequencing ladders, we can tell exactly where the protein bound. T C G G A G C A A C G C A A A C A A A C G T G C T T G G * Topic 4: Determining the Structure of Protein and nucleic acids CHAPTER20: Techniques of Molecular Biology X-ray crystallography (X-晶体衍射) NMR (核磁共振) * X-ray crystallography