electrophoretic mobility shift assay - wikipedia, the free encyclopedia
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Lane 1 is a negative control, andcontains only genetic material. Lane 2contains protein as well as a DNAfragment that, based on its sequence,does not interact. Lane 3 containsprotein and a DNA fragment thatdoes react; the resulting complex islarger, heavier, and slower-moving.The pattern shown in lane 3 is the onethat would result if all the DNA werebound and no dissociation of complexoccurred during electrophoresis.When these conditions are not met asecond band might be seen in lane 3reflecting the presence of free DNAor the dissociation of theDNA-protein complex.
Electrophoretic mobility shift assayFrom Wikipedia, the free encyclopedia
An electrophoretic mobility shift assay (EMSA) or mobility shiftelectrophoresis, also referred as a gel shift assay, gel mobility shiftassay, band shift assay, or gel retardation assay, is a common affinityelectrophoresis technique used to study protein–DNA or protein–RNAinteractions. This procedure can determine if a protein or mixture ofproteins is capable of binding to a given DNA or RNA sequence, andcan sometimes indicate if more than one protein molecule is involved inthe binding complex. Gel shift assays are often performed in vitroconcurrently with DNase footprinting, primer extension, andpromoter-probe experiments when studying transcription initiation,DNA replication, DNA repair or RNA processing and maturation.Although precursors can be found in earlier literature, most currentassays are based on methods described by Garner and Revzin [1] andFried and Crothers.[2]
Principle
A mobility shift assay is electrophoretic separation of a protein–DNA orprotein–RNA mixture on a polyacrylamide or agarose gel for a shortperiod (about 1.5-2 hr for a 15- to 20-cm gel).[3] The speed at whichdifferent molecules (and combinations thereof) move through the gel isdetermined by their size and charge, and to a lesser extent, their shape(see gel electrophoresis). The control lane (DNA probe without proteinpresent) will contain a single band corresponding to the unbound DNAor RNA fragment. However, assuming that the protein is capable ofbinding to the fragment, the lane with protein present will containanother band that represents the larger, less mobile complex of nucleicacid 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 theratio of bound to unbound nucleic acid on the gel reflects the fraction of free and bound probe molecules as thebinding reaction enters the gel. This stability is in part due to the low ionic strength of the buffer, but also due toa "caging effect", in that the protein, surrounded by the gel matrix, is unable to diffuse away from the probebefore they recombine. If the starting concentrations of protein and probe are known, and if the stoichiometry ofthe complex is known, the apparent affinity of the protein for the nucleic acid sequence may be determined. Thenumber derived is the apparent Kd as the true Kd cannot be determined from this type of assay because there isno equilibrium binding between the substrate complexes. If the protein concentration is not known but thecomplex stoichiometry is, the protein concentration can be determined by increasing the concentration of DNAprobe until further increments do not increase the fraction of protein bound. By comparison with a set ofstandard 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 agreater shift. This method is referred to as a supershift assay, and is used to unambiguously identify a proteinpresent in the protein – nucleic acid complex.
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Often, an extra lane is run with a competitor oligonucleotide to determine the most favorable binding sequencefor the binding protein. The use of different oligonucleotides of defined sequence allows the identification ofthe precise binding site by competition (not shown in diagram). Variants of the competition assay are useful formeasuring 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 foridentification of the transcription factor. Consensus sequence oligonucleotides for the transcription factor ofinterest will be able to compete for the binding, eliminating the shifted band, and must be confirmed bysupershift. If the predicted consensus sequence fails to compete for binding, identification of the transcriptionfactor may be aided by Multiplexed Competitor EMSA (MC-EMSA), whereby large sets of consensussequences are multiplexed in each reaction, and where one set competes for binding, the individual consensussequences from this set are run in a further reaction.[4]
For visualization purposes, the nucleic acid fragment is usually labelled with a radioactive, fluorescent or biotinlabel. Standard ethidium bromide staining is less sensitive than these methods and can lack the sensitivity todetect the nucleic acid if small amounts are used in these experiments. When using a biotin label, streptavidinconjugated to an enzyme such as horseradish peroxidase is used to detect the DNA fragment (Non-radioactiveEMSA review (http://www.biocompare.com/review/738/LightShift-Chemiluminescent-EMSA-Kit-From-Pierce.html) ). While isotopic DNA labeling has little or no effect on protein binding affinity, use ofnon-isotopic labels including flurophores or biotin can alter the affinity and/or stoichiometry of the proteininteraction of interest. Competition between fluorophore- or biotin-labeled probe and unlabeled DNA of thesame sequence can be used to determine whether the label alters binding affinity or stoichiometry.
References^ Garner MM, Revzin A (July 1981). "A gel electrophoresis method for quantifying the binding of proteins tospecific DNA regions: application to components of the Escherichia coli lactose operon regulatory system"(http://nar.oxfordjournals.org/cgi/pmidlookup?view=long&pmid=6269071) . Nucleic Acids Res. 9 (13): 3047–60.doi:10.1093/nar/9.13.3047 (http://dx.doi.org/10.1093%2Fnar%2F9.13.3047) . PMC 327330 (//www.ncbi.nlm.nih.gov/pmc/articles/PMC327330) . PMID 6269071 (//www.ncbi.nlm.nih.gov/pubmed/6269071) .http://nar.oxfordjournals.org/cgi/pmidlookup?view=long&pmid=6269071.
1.
^ Fried M, Crothers DM (December 1981). "Equilibria and kinetics of lac repressor-operator interactions bypolyacrylamide gel electrophoresis" (http://nar.oxfordjournals.org/cgi/pmidlookup?view=long&pmid=6275366) .Nucleic Acids Res. 9 (23): 6505–25. doi:10.1093/nar/9.23.6505 (http://dx.doi.org/10.1093%2Fnar%2F9.23.6505) .PMC 327619 (//www.ncbi.nlm.nih.gov/pmc/articles/PMC327619) . PMID 6275366 (//www.ncbi.nlm.nih.gov/pubmed/6275366) . http://nar.oxfordjournals.org/cgi/pmidlookup?view=long&pmid=6275366.
2.
^ a b Ausubel, Frederick M. (1994). Current Protocols in molecular biology. Chichester: John Wiley & Sons.pp. 12.2.1–11. ISBN 0-471-50337-1.
3.
^ Smith AJ, Humphries SE (January 2009). "Characterization of DNA-binding proteins using multiplexed competitorEMSA". J. Mol. Biol. 385 (3): 714–7. doi:10.1016/j.jmb.2008.11.035 (http://dx.doi.org/10.1016%2Fj.jmb.2008.11.035) . PMID 19059416 (//www.ncbi.nlm.nih.gov/pubmed/19059416) .
4.
Protocols
Dr. Mirmira EMSA Protocol (http://protocolpedia.com/index.php?option=com_sobi2&sobi2Task=sobi2Details&sobi2Id=52&Itemid=81)Preparation of nuclear extract for EMSA (http://protocolpedia.com/index.php?option=com_sobi2&sobi2Task=sobi2Details&sobi2Id=352&Itemid=81)Jonathan Flint Lab Protocol (http://www.well.ox.ac.uk/flint/EMSA.htm)EMSA for Transcription Factor Binding (http://www.protocol-online.org/prot/Protocols/Gel-Mobility-
Electrophoretic mobility shift assay - Wikipedia, the free encyclopedia http://en.wikipedia.org/wiki/Electrophoretic_mobility_shift_assay
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Shift-Assay-for-transcription-factor-binding-2840.html)Chemiluminescent Gel Shift Protocol (http://www.activemotif.com/documents/1680.pdf)
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