Electrophoretic Mobility Shift Assay
The electrophoretic mobility shift assay (EMSA ) or band shift assay is an affinity electrophoresis and used for the detection of DNA or RNA -binding proteins, such as transcription factors.
Here, proteins are incubated with a DNA fragment of known sequence. In the DNA sequence, there are usually a regulatory region of a gene (eg, a promoter or enhancer ). The sample is applied to a polyacrylamide gel and an electric field by means of the protein complexes, and DNA may be an antibody can be separated according to their size. Compared to the pure protein or DNA band appears in the gel electrophoresis run wide shift ( engl. ribbon shift) in accordance with the load, conformation and size of the protein -ligand complex. DNA-protein -antibody complexes migrate more slowly than the DNA -protein complexes which migrate more slowly than unbound DNA, unbound proteins migrate fastest. By dilution series to Ks values can be determined.
By labeling of the DNA ( for example with a radionuclide, by digoxygenin - labeling or using a fluorescent dye ), the bands in the gel can be visualized. The less a complex migrated in the gel, the greater is its molar mass. This is used to specify not only the DNA of the bands and the antibody, but may also be non- labeled DNA in different amounts of blocking as well as mutated DNA can be used for filtering of a specific signal.
Contains Affinitäselektrophorese addition of DNA and an interacting protein or an antibody against the protein, the assay is referred to as a super shift assay.