Electrophoretic mobility shift assay

This stability is in part due to a "caging effect", in that the protein, surrounded by the gel matrix, is unable to diffuse away from the probe before they recombine.

[6] Unless the complex is very long lived under gel conditions, or dissociation during electrophoresis is taken into account, the number derived is an apparent Kd.

[citation needed] Once DNA-protein binding is determined in vitro, a number of algorithms can narrow the search for identification of the transcription factor.

Consensus sequence oligonucleotides for the transcription factor of interest will be able to compete for the binding, eliminating the shifted band, and must be confirmed by supershift.

Competition between fluorophore- or biotin-labeled probe and unlabeled DNA of the same sequence can be used to determine whether the label alters binding affinity or stoichiometry.

Lane 1 is a negative control, and contains only genetic material. 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. The pattern shown in lane 3 is the one that would result if all the DNA were bound and no dissociation of complex occurred during electrophoresis. When these conditions are not met a second band might be seen in lane 3 reflecting the presence of free DNA or the dissociation of the DNA-protein complex.
Schematic of an EMSA showing a gel shift in response to more protein
Schematic of an EMSA showing the labeled DNA (dark band) move up the gel as more protein is added, as indicted by the triangle below