Electromobility Shift Assay (EMSA)
The electromobility shift assay (EMSA), aka gel retardation assay, assesses protein-DNA or protein RNA interactions.
Basic Principle
The basic principle is quite simple. A protein-DNA (or RNA) mixture is resolved through an agarose gel by electrophoresis. The speed at which different molecules migrate through the gel is determined by their size and charge, and to a lesser extent, their shape. The negative control lane contains DNA probe without protein present, and will appear as a single band corresponding to the unbound DNA or RNA fragment. A lane with DNA and 'bound" protein will appear as a larger sized band which appears to have "shifted" higher up in the gel in reference to the negative control lane. This "shift" corresponds to a larger, less mobile complex of nucleic acid probe bound to protein.
Visualization
For visualization, the nucleic acid fragment is usually labelled with a radioactive, fluorescent or biotin label. Ethidium bromide staining may lack the sensitivity required 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
Additional parameters
The EMSA can be utilized to determine more than a binary "yes" or "no" answer with respect to protein-nucleic acid interaction. 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. The identity of a bound protein can be determined by introducing an antibody into the protein-DNA mixture. An antibody that recognizes the protein creates 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.
For any protein-nucleic acid complex, the binding sequence on the nucleic acid can be determined by introducing different oligonucleotides of defined sequence into the mixture. This allows for identification of the precise binding site by competition. Variants of the competition assay are useful for measuring the specificity of binding and for measurement of association and dissociation kinetics.
Once DNA-protein binding is determined in vitro, a number of in silico algorithms can narrow the search for identification of the transcription factor. Consensus sequence oligonucleotides for the transcription factor of interest will be able to compete for the binding, eliminating the shifted band, and must be confirmed by supershift. If the predicted consensus sequence fails to compete for binding, identification of the transcription factor may be aided by Multiplexed Competitor EMSA (MC-EMSA), whereby large sets of consensus sequences are multiplexed in each reaction, and where one set competes for binding, the individual consensus sequences from this set are run in a further reaction.
Basic Principle
The basic principle is quite simple. A protein-DNA (or RNA) mixture is resolved through an agarose gel by electrophoresis. The speed at which different molecules migrate through the gel is determined by their size and charge, and to a lesser extent, their shape. The negative control lane contains DNA probe without protein present, and will appear as a single band corresponding to the unbound DNA or RNA fragment. A lane with DNA and 'bound" protein will appear as a larger sized band which appears to have "shifted" higher up in the gel in reference to the negative control lane. This "shift" corresponds to a larger, less mobile complex of nucleic acid probe bound to protein.
Visualization
For visualization, the nucleic acid fragment is usually labelled with a radioactive, fluorescent or biotin label. Ethidium bromide staining may lack the sensitivity required 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
Additional parameters
The EMSA can be utilized to determine more than a binary "yes" or "no" answer with respect to protein-nucleic acid interaction. 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. The identity of a bound protein can be determined by introducing an antibody into the protein-DNA mixture. An antibody that recognizes the protein creates 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.
For any protein-nucleic acid complex, the binding sequence on the nucleic acid can be determined by introducing different oligonucleotides of defined sequence into the mixture. This allows for identification of the precise binding site by competition. Variants of the competition assay are useful for measuring the specificity of binding and for measurement of association and dissociation kinetics.
Once DNA-protein binding is determined in vitro, a number of in silico algorithms can narrow the search for identification of the transcription factor. Consensus sequence oligonucleotides for the transcription factor of interest will be able to compete for the binding, eliminating the shifted band, and must be confirmed by supershift. If the predicted consensus sequence fails to compete for binding, identification of the transcription factor may be aided by Multiplexed Competitor EMSA (MC-EMSA), whereby large sets of consensus sequences are multiplexed in each reaction, and where one set competes for binding, the individual consensus sequences from this set are run in a further reaction.