DNA Retardation Gels (6%), 1.0 mm

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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The Invitrogen 6% DNA Retardation Gels contain 0.5X TBE. Both gels will work for gel retardation; however, the 1X TBE in the Invitrogen 6% TBE Gels have a higher ionic environment, which may affect DNA-protein interactions. The 0.5X TBE used in the Invitrogen 6% DNA Retardation Gels usually works better, as it offers good fragment separation in electrophoresis yet has an ionic strength low enough to promote DNA-protein interactions.

Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.

The 6% DNA retardation gels are 0.5X TBE. Both gels will work for gel mobility shift assays, but the 1X TBE has a higher ionic environment that may affect DNA/protein interactions. 0.5X TBE usually works better.

Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.