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If a regulatory protein binds to a segment of DNA it will slow the migration of the DNA through a gel.

teins bind, although others may be involved in bending DNA or forming stem and loop structures in either the DNA itself or the RNA. After sequencing a gene and its regulatory region, the sequence may be analyzed for known protein binding motifs. However, many cases are known of computer predicted binding sites that do not actually function in real life. Deletion analysis of these upstream sites determines how they affect gene expression, but do not show if a protein actually binds to the site. Consequently, even after a presumed binding site has been found, the binding of the regulatory protein must be confirmed experimentally.

One approach to deciding if a particular regulatory protein affects gene expression is to test directly if the protein binds to DNA from the upstream region. To do this we need both purified DNA from the regulatory region and also purified regulatory protein. The mobility of a fragment of DNA in a gel is altered upon binding protein. This procedure is therefore known either as an electrophoretic mobility shift assay, bandshift assay or gel retardation assay (Fig. 25.10).

bandshift assay Method for testing binding of a protein to DNA by measuring the change in mobility of DNA during gel electrophoresis. Same as gel retardation or mobility shift assay gel retardation Method for testing binding of a protein to DNA by measuring the change in mobility of DNA during gel electrophoresis. Same as bandshift assay or mobility shift assay mobility shift assay Method for testing binding of a protein to DNA by measuring the change in mobility of DNA during gel electrophoresis. Same as bandshift assay or gel retardation

A) DNA TO BE ANALYZED

Regulatory region Coding region a ยก b ! c ! d ! e^=DNA

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Cut site Cut site Cut site Cut site

Cut with restriction enzyme

No regulatory Regulatory

PROTEIN ADDED PROTEIN ADDED

B) Agarose gel

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