Band is retarded f f
No protein With added regulatory protein
FIGURE 25.10 Gel Retardation to Assess Protein Binding to DNA
Gel retardation assays determine whether or not a specific regulatory protein actually binds to the DNA in the regulatory region of a gene. The regulatory and coding regions of a gene are cut into various fragments by a restriction enzyme. The fragments are divided into two samples. In one (I), no regulatory protein is added, whereas in the other sample (II), purified regulatory protein is mixed with the DNA. The DNA fragments are separated by size using agarose gel electrophoresis. If the regulatory protein binds to the DNA, that fragment is heavier and travels slower. It is therefore retarded relative to its position in the absence of protein. In this example, fragment c has a binding site for the regulatory protein and its band is retarded.
FIGURE 25.11 Footprint Analysis—Procedure
To identify the exact location of a protein binding site, the fragment that contains the binding site is first isolated. The fragment is mixed with the protein, and then treated with DNase. This enzyme cuts randomly along the length of the DNA fragment, but will not be able to cut in the region where the protein binds.
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