凝胶阻滞.ppt

凝胶阻滞;;;;Figure 7-29. A gel-mobility shift assay. The principle of the assay is shown schematically in (A). In this example an extract of an antibody-producing cell line is mixed with a radioactive DNA fragment containing about 160 nucleotides of a regulatory DNA sequence from a gene encoding the light chain of the antibody made by the cell line. The effect of the proteins in the extract on the mobility of the DNA fragment is analyzed by polyacrylamide-gel electrophoresis followed by autoradiography. The free DNA fragments migrate rapidly to the bottom of the gel, while those fragments bound to proteins are retarded; the finding of six retarded bands suggests that the extract contains six different sequence-specific DNA-binding proteins (indicated as C1–C6) that bind to this DNA sequence. (For simplicity, any DNA fragments with more than one protein bound have been omitted from the figure.) In (B) the extract was fractionated by a standard chromatographic technique (top), and each fraction was mixed with the radioactive DNA fragment, applied to one lane of a polyacrylamide gel, and analyzed as in (A). (B, modified from C. Scheidereit, A. Heguy, and R.G. Roeder, Cell 51:783–793, 1987.) ;Figure 7-30. DNA affinity chromatography. In the first step, all the proteins that can bind DNA are separated from the remainder of the cellular proteins on a column containing a huge number of different DNA sequences. Most sequence-specific DNA-binding proteins have a weak (nonspecific) affinity for bulk DNA and are therefore retained on the column. This affinity is due largely to ionic attractions, and the proteins can be washed off the DNA by a solution that contains a moderate concentration of salt. In the second step, the mixture of DNA-binding proteins is passed through a column that contains only DNA of a particular sequence. Typically, all the DNA-binding proteins will stick to the column, the great majority by nonspecific interactions. These are again eluted by solutions of mo