Electrophoretic mobility shift assay

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Lane 1 is a negative control, and contains only DNA. Lane 2 contains protein as well as a DNA fragment that, based on its sequence, does not interact. Lane 3 contains protein and a DNA fragment that does react; the resulting complex is larger, heavier, and slower-moving. This situation is contrived, and in reality there would likely be a second band in lane 3 owing to dissociation of the DNA-protein complex.

An electrophoretic mobility shift assay (EMSA), also referred as a gel shift assay or band shift assay or gel retardation assay , is a common technique used to study protein-DNA or protein-RNA interactions. This procedure can determine if a protein or mixture of proteins is capable of binding to a given DNA or RNA sequence, and can sometimes indicate if more than one protein molecule is involved in the binding complex. Gel shift assays are often performed in vitro concurrently with DNase footprinting, primer extension, and promoter-probe experiments when studying transcription initiation.

[edit] Principle

A mobility shift assay generally involves electrophoretic separation of a protein-DNA mixture on a polyacrylamide or agarose gel for a short period. The speed at which different molecules (and combinations thereof) move through the gel is determined by their size and charge, and to a lesser extent, their shape (see Gel electrophoresis). The control lane without protein present will contain a single band corresponding to the unbound DNA fragment. However, assuming the protein is capable of binding to the DNA fragment, the lane with protein present will contain another band that represents the larger complex of DNA bound to protein. From the ratio of bound to unbound DNA, the affinity of the protein to the DNA sequence may be determined.

Often, an extra lane is run with a competitor oligonucleotide to determine the most favorable binding sequence for the binding protein. The use of different oligonucleotides of defined sequence allows the identification of the precise binding site by competition. (Not shown in diagram). Variants of the competition assay are useful for measuring the specificity of binding and for measurement of association and dissociation kinetics.

For visualization purposes, the DNA fragment is usually labeled with a radioactive or fluorescent label, as standard ethidium bromide staining lacks the sensitivity to detect the relatively small amounts of DNA used in these experiments.