Electrophoretic Mobility Shift Assay

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Lane 1 contains only DNA as a negative control , lane 2 contains DNA and a non-binding protein, lane 3 contains DNA and a binding protein. If the binding is incomplete, an additional band of unbound DNA can appear.

The electrophoretic mobility shift assay ( EMSA ) or band shift assay is an affinity electrophoresis and used for the detection of DNA - or RNA -binding proteins , such as transcription factors .

properties

To determine protein-DNA interactions using EMSA, proteins are incubated with a DNA fragment of known sequence, in the case of protein-RNA interactions, correspondingly with an RNA fragment. The DNA sequence is usually a regulatory area of a gene (for example a promoter or enhancer ). The sample is applied to an agarose or polyacrylamide gel and the complexes of protein and DNA are separated according to their size using an electric field. Compared to the pure DNA band, a band shift occurs during gel electrophoresis , depending on the charge, conformation and size of the protein-ligand complex. If affinity electrophoresis contains DNA and an interacting protein as well as an antibody against the protein, the assay is known as a supershift assay . DNA-protein-antibody complexes migrate more slowly than DNA-protein complexes, which migrate more slowly than unbound DNA. In contrast to DNA- and RNA-binding proteins , which often have a positive net charge, unbound acidic proteins can also enter the gel in the buffer system used . However, since the proteins are not marked, they will not be visible without a protein color such as B. a silver color . By dilution series can be affinities determined. The complexes do not dissociate in the electrophoresis due to a cage effect of the pores in the gel.

By labeling the DNA, the bands in the gel can be made visible, for example by labeling molecules with a radionuclide , digoxygenin labeling, biotinylation or by means of fluorescent labeling . In this case, not only the DNA and the antibodies are used to specify the bands, but also non-labeled DNA can be used in different amounts for blocking, but also mutated DNA can be used to filter out a specific signal. The use of point mutants in the labeled DNA or in the unlabeled competitor allows conclusions to be drawn about the nucleotides that are important for the binding.

The EMSA, which, due to its uncomplicated application, is one of the common methods used to elucidate the mechanisms of gene regulation, is based on the work of Garner and Revzin and Fried and Crothers.

A modification is the methylation and uracil interference assay, in which the influence of modified bases on DNA binding is examined. It allows conclusions to be drawn about the nucleotides that are involved in the protein-DNA interaction.

literature

  • Tom Moss (Editor): DNA'Protein Interactions: Principles and Protocols (Methods in Molecular Biology) , Humana, 2nd Edition 2001, ISBN 978-0-89603-671-0 .

Web links

Individual evidence

  1. ↑ a b Garner MM, Revzin A: 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 . In: Nucleic Acids Research . 9, No. 13, July 1981, pp. 3047-3060. doi : 10.1093 / nar / 9.13.3047 . PMID 6269071 . PMC 327330 (free full text).
  2. ^ A b Fried M, Crothers DM: Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis . In: Nucleic Acids Research . 9, No. 23, December 1981, pp. 6505-6525. doi : 10.1093 / nar / 9.23.6505 . PMID 6275366 . PMC 327619 (free full text).
  3. ^ Ausubel, Frederick M .: Current Protocols in molecular biology . John Wiley & Sons, Chichester 1994, ISBN 0-471-50337-1 , pp. 12.2.1-11.
  4. ↑ Fried MG (1989) Measurement of protein-DNA interaction parameters by electrophoresis mobility shift assay. In: Electrophoresis 10, 366-376. PMID 2670548 .
  5. ^ Fried MG, Liu G. (1994). Molecular sequestration stabilizes CAP-DNA complexes during polyacrylamide gel electrophoresis. In: Nucleic Acids Research 22 (23): 5054-5059. doi: 10.1093 / nar / 22.23.5054 . PMID 7800499 .
  6. ↑ Jiří Kozelka: Evaluation of dissociation constants from competition binding experiments based on the relative binding ratio . In: Analytical Biochemistry . 409, No. 1, 2011, ISSN  0003-2697 , pp. 66-73. doi : 10.1016 / year from 2010.09.023 .
  7. ^ Albert S. Baldwin, Marjorie Oettinger, Kevin Struhl: Methylation and Uracil Interference Assays for Analysis of Protein-DNA Interactions . In: Current Protocols in Molecular Biology . 12, 2001. doi : 10.1002 / 0471142727.mb1203s36 .