Electrophoretic mobility shift assay
An electrophoretic mobility shift assay (EMSA), also referred as a gel shift assay, gel mobility shift assay, band shift assay, or gel retardation assay, is a common technique used to study protein-DNA or protein-RNA interactions. This procedure can determine if a protein or mixture of proteins is capable of binding to a given DNA or RNA sequence, and can sometimes indicate if more than one protein molecule is involved in the binding complex. Gel shift assays are often performed in vitro concurrently with DNase footprinting, primer extension, and promoter-probe experiments when studying transcription initiation, DNA replication, DNA repair or RNA processing and maturation. Although precursors can be found in earlier literature, most current assays are based on methods described by Garner and Revzin [1] and Fried and Crothers [2].
Principle
A mobility shift assay generally involves electrophoretic separation of a protein-DNA or protein-RNA mixture on a polyacrylamide or agarose gel for a short period (about 1.5-2 hr for a 15- to 20-cm gel). [3] 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 (see gel electrophoresis). The control lane (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).
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", in that 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, the affinity of the protein for the nucleic acid sequence may be determined. If the protein concentration is not known, it can be determined by increasing the concentration of DNA probe until further increments do not increase the fraction of protein bound. By comparison with a set of standard dilutions of free probe run on the same gel, the number of moles of protein can be calculated. [3]
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.
Often, an extra lane is run with a competitor oligonucleotide to determine the most favorable binding sequence for the binding protein. The use of different oligonucleotides of defined sequence allows the identification of the precise binding site by competition (not shown in diagram). Variants of the competition assay are useful for measuring the specificity of binding and for measurement of association and dissociation kinetics.
For visualization purposes, the nucleic acid fragment is usually labeled with a radioactive, fluorescent or biotin label. Standard ethidium bromide staining is less sensitive than these methods and can lack the sensitivity to detect the nucleic acid if small amounts are used in these experiments. When using a biotin label, streptavidin conjugated to an enzyme such as horseradish peroxidase is used to detect the DNA fragment (Non-radioactive EMSA review).
References
- ↑ Garner, M.M. and Revzin, A. (1981) "A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system." Nucleic Acids Res. 9:3047-3060. [1]
- ↑ Fried, M. and Crothers, D.M. (1981) "Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis." Nucleic Acids Res., 9:6505-6525. [2]
- ↑ 3.0 3.1 Ausubel, Frederick M. (1994). Current protocols in molecular biology. Chichester: John Wiley & Sons. pp. 12.2.1-12.2.11. ISBN 0-471-50337-1.