Electrophoretic Mobility-Shift Assay (EMSA) Kit, with SYBR™ Green & SYPRO™ Ruby EMSA stains - FAQs

A supershift assay is a method for positively identifying a protein:DNA interaction on an EMSA. An antibody (typically 1 µg) is added to the binding reaction. During electrophoresis, the antibody:protein:DNA complex migrates slowly, causing a “supershift” compared to the “shift” caused by a protein:DNA complex. Not all antibodies will cause a supershift. Some antibodies do not bind to proteins once they are bound to DNA. Some antibodies can prevent protein:DNA interactions but can still be used to confirm the identity of a protein that causes a shift in the absence of the antibody.

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EMSAs (also called gel shifts, band shifts, gel retardation assays, or mobility assays) have been used extensively for studying protein-DNA interactions. Because protein-DNA complexes migrate more slowly through a native polyacrylamide or agarose gel than DNA alone, individual protein-DNA complexes can be visualized as discrete bands within the gel using chemiluminescence or radioisotopic detection.

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A shift assay is a DNA-binding assay using nondenaturing PAGE. It provides a simple, rapid, and extremely sensitive method for detecting sequence-specific DNA-binding proteins. Proteins bind specifically to an end-labeled DNA fragment corresponding to the individual protein-DNA complexes. You can use the assay to test binding of purified proteins or of uncharacterized factors in crude extracts. This assay also permits quantitative determination of the affinity, abundance, association rate constants, dissociation rate constants, and binding specificity of DNA-binding proteins.

A supershift assay is a variation of the mobility shift DNA-binding assay that uses antibodies to identify proteins present in the protein-DNA complex.Addition of a specific antibody to a binding reaction can have one of several effects. If the protein recognized by the antibody is not involved in complex formation, addition of the antibody should have no effect. If the protein that forms the complex is recognized by the antibody, the antibody can either block complex formation or it can form an antibody-protein-DNA ternary complex and thereby specifically result in a further reduction in the mobility of the protein-DNA complex (a supershift). Results may be different depending upon whether the antibody is added before or after the protein binds DNA (particularly if there are epitopes on the DNA binding surface of the protein).

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Here is the composition of the 5X Binding Buffer:

- 50 mM Tris HCl, pH 7.4
- 750 mM KCl
- 0.5 mM DTT
- 0.5 mM EDTA

Note: This buffer is optimized for lac repressor binding to lac operator. It may not be optimal for other protein-nucleic acid interactions.

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