Electrophoretic mobility shift assay
Encyclopedia
An electrophoretic mobility shift assay (EMSA) or mobility shift electrophoresis, also referred as a gel shift assay, gel mobility shift assay, band shift assay, or gel retardation assay, is a common affinity electrophoresis
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 and Fried and Crothers.
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). 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.
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.
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.
For visualization purposes, the nucleic acid fragment is usually labelled 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).
Affinity electrophoresis
Affinity electrophoresis is a general name for many analytical methods used in biochemistry and biotechnology. Both qualitative and quantitative information may be obtained through affinity electrophoresis. The methods include the so-called mobility shift electrophoresis, charge shift...
technique used to study protein–DNA or protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
–RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
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
In vitro
In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...
concurrently with DNase footprinting, primer extension
Primer extension
Primer extension is a technique whereby the 5' ends of RNA or DNA can be mapped.Primer extension can be used to determine the start site of RNA transcription for a known gene. This technique requires a radiolabelled primer which is complementary to a region near the 5' end of the gene...
, and promoter-probe experiments when studying transcription
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
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 and Fried and Crothers.
Principle
A mobility shift assay is electrophoretic separationGel electrophoresis
Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...
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). 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
Gel electrophoresis
Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...
). 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.
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
Oligonucleotide
An oligonucleotide is a short nucleic acid polymer, typically with fifty or fewer bases. Although they can be formed by bond cleavage of longer segments, they are now more commonly synthesized, in a sequence-specific manner, from individual nucleoside phosphoramidites...
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.
Once DNA-protein binding is determined in vitro, a number of in silico
In silico
In silico is an expression used to mean "performed on computer or via computer simulation." The phrase was coined in 1989 as an analogy to the Latin phrases in vivo and in vitro which are commonly used in biology and refer to experiments done in living organisms and outside of living organisms,...
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.
For visualization purposes, the nucleic acid fragment is usually labelled with a radioactive
Isotopic labeling
Isotopic labeling is a technique for tracking the passage of a sample of substance through a system. The substance is 'labeled' by including unusual isotopes in its chemical composition...
, fluorescent or biotin
Biotinylation
In biochemistry, biotinylation is the process of covalently attaching biotin to a protein, nucleic acid or other molecule. Biotinylation is rapid, specific and is unlikely to perturb the natural function of the molecule due to the small size of biotin...
label. Standard ethidium bromide
Ethidium bromide
Ethidium bromide is an intercalating agent commonly used as a fluorescent tag in molecular biology laboratories for techniques such as agarose gel electrophoresis. It is commonly abbreviated as "EtBr", which is also an abbreviation for bromoethane...
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
Streptavidin
Streptavidin is a 60000 dalton protein purified from the bacterium Streptomyces avidinii. Streptavidin homo-tetramers have an extraordinarily high affinity for biotin . With a dissociation constant on the order of ≈10-14 mol/L, the binding of biotin to streptavidin is one of the strongest...
conjugated to an enzyme such as horseradish peroxidase is used to detect the DNA fragment (Non-radioactive EMSA review).