7.25E: DNA Mobility Shifts

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DNA mobility shift assay is a technique for studying gene regulation and determining protein-DNA interactions.

Learning Objectives

Identify the utility of DNA mobility shift assays

Key Points

The interaction of proteins with DNA is central to the control of many cellular processes, including DNA replication, recombination and repair, transcription, and viral assembly.
An advantage of studying protein-DNA interactions by an electrophoretic mobility shift assay is the ability to resolve complexes of different stoichiometry or conformation.
The source of the DNA-binding protein may be a crude nuclear or whole cell extract, in vitro transcription product, or a purified preparation.

Key Terms

polyacrylamide: Any of a range of cross-linked polymers of acrylamide; used to form soft gels.

A mobility shift assay is electrophoretic separation of a protein-DNA or protein- RNA 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. The control lane (a DNA probe without protein present) will contain a single band corresponding to the unbound DNA or RNA fragment. However, assuming that the protein is capable of binding to the fragment, the lane with protein present will contain another band that represents the larger, less mobile, complex of nucleic acid probe bound to protein, which is “shifted” up on the gel (since it has moved more slowly).

Figure: **Gel Shift Assay**: 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. 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.

Under the correct experimental conditions, the interaction between the DNA and protein is stabilized and the ratio of bound to unbound nucleic acid on the gel reflects the fraction of free and bound probe molecules as the binding reaction enters the gel. This stability is in part due to the low ionic strength of the buffer, but also due to a “caging effect”; the protein, surrounded by the gel matrix, is unable to diffuse away from the probe before they recombine. If the starting concentrations of protein and probe are known, and if the stoichiometry of the complex is known, the apparent affinity of the protein for the nucleic acid sequence may be determined. An antibody that recognizes the protein can be added to this mixture to create an even larger complex with a greater shift. This method is referred to as a supershift assay, and is used to unambiguously identify a protein present in the protein-nucleic acid complex.