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- DNAor protein- RNAinteractions. 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 vitroconcurrently with DNase footprinting, primer extension, and promoter-probeexperiments 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 [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. [http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=6269071] ] and Fried and Crothers [ 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. [http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=6275366] ] .
A mobility shift assay is electrophoretic separation of a protein-DNA or protein-RNA mixture on a polyacrylamide or
agarose gelfor a short period (about 1.5-2 hr for a 15- to 20-cm gel). cite book |author=Ausubel, Frederick M. |title=Current protocols in molecular biology |publisher=John Wiley & Sons |location=Chichester |year=1994 |pages=12.2.1-12.2.11 |isbn=0-471-50337-1 |oclc= |doi=] 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
oligonucleotideto 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 bromidestaining 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, streptavidinconjugated to an enzyme such as horseradish peroxidase is used to detect the DNA fragment ( [http://www.biocompare.com/review/738/LightShift-Chemiluminescent-EMSA-Kit-From-Pierce.html Non-radioactive EMSA review] ).
* [http://www.well.ox.ac.uk/flint/EMSA.htm Jonathan Flint Lab Protocol]
* [http://www.protocol-online.org/prot/Protocols/Gel-Mobility-Shift-Assay-for-transcription-factor-binding-2840.html EMSA for Transcription Factor Binding]
Wikimedia Foundation. 2010.
Look at other dictionaries:
Electrophoretic Mobility Shift Assay — Der Electrophoretic Mobility Shift Assay (EMSA) oder Band Shift Assay dient zum Nachweis von DNA oder RNA bindenden Proteinen, beispielsweise Transkriptionsfaktoren. Weitere Möglichkeiten bestehen darin, Protein Liganden Interaktionen jeglicher… … Deutsch Wikipedia
Electrophoretic Mobility Shift Assay — Retard sur gel Pour les articles homonymes, voir EMSA. Le retard sur gel (EMSA ou electrophoretic mobility shift assay) est une technique de biologie moléculaire permettant de détecter une interaction entre une protéine et de l ADN ou de l ARN.… … Wikipédia en Français
electrophoretic mobility shift assay — gel retardation a … Medical dictionary
Assay — Das Wort Assay (deutsch: Test, Probe) stammt aus dem englischen Sprachraum und bedeutet dort allgemein eine Untersuchung zum Nachweis bestimmter Substanzen (z. B. Metalle). In der deutschen Sprache bezeichnet Assay vor allem in der… … Deutsch Wikipedia
gel retardation assay — (= mobility shift assay) Test for interaction between molecules by looking for a change in gel electrophoretic mobility. For example, to assay for levels of a transcription factor, cell extracts are incubated with a radiolabelled oligonucleotide… … Dictionary of molecular biology
EMSA — electrophoretic mobility shift assay; Emergency Medical Services Agency … Medical dictionary
EMSA — • electrophoretic mobility shift assay; • Emergency Medical Services Agency … Dictionary of medical acronyms & abbreviations
Gel electrophoresis — apparatus – An agarose gel is placed in this buffer filled box and electrical field is applied via the power supply to the rear. The negative terminal is at the far end (black wire), so DNA migrates toward the camera. Classification… … Wikipedia
GSA — steht für: Gel Shift Assay, ein Verfahren zum Nachweis von DNA oder RNA bindenden Proteinen, siehe Electrophoretic Mobility Shift Assay General Services Administration, eine unabhängige Agentur der US Regierung für amerikanische Bundesbehörden… … Deutsch Wikipedia
DNA binding site — DNA binding sites are a type of binding site found in DNA where other molecules may bind. DNA binding sites are distinct from other binding sites in that (1) they are part of a DNA sequence (e.g. a genome) and (2) they are bound by DNA binding… … Wikipedia