Standard in vitro assays for protein-nucleic acid interactions - Gel shift assays for RNA and DNA binding

Sarah F. Mitchell, Jon R. Lorsch

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

The characterization of protein-nucleic acid interactions is necessary for the study of a wide variety of biological processes. One straightforward and widely used approach to this problem is the electrophoretic mobility shift assay (EMSA), in which the binding of a nucleic acid to one or more proteins changes its mobility through a nondenaturing gel matrix. Usually, the mobility of the nucleic acid is reduced, but examples of increased mobility do exist. This type of assay can be used to investigate the affinity of the interaction between the protein and nucleic acid, the specificity of the interaction, the minimal binding site, and the kinetics of the interaction. One particular advantage of EMSA is the ability to analyze multiple proteins, or protein complexes, binding to nucleic acids. This assay is relatively quick and easy and utilizes equipment available in most laboratories; however, there are many variables that can only be determined empirically; therefore, optimization is necessary and can be highly dependent upon the system. The protocol described here is for the poly(A)-binding protein (PABP) binding to an unstructured RNA probe of 43 bases. While this may be a useful protocol for some additional assays, it is recommended that both reaction conditions and gel running conditions be tailored to the individual interaction to be probed.

Original languageEnglish (US)
Title of host publicationLaboratory Methods in Enzymology
Subtitle of host publicationProtein Part C
PublisherAcademic Press Inc.
Pages179-196
Number of pages18
ISBN (Print)9780124201194
DOIs
StatePublished - 2014

Publication series

NameMethods in Enzymology
Volume541
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • Coomassie blue staining
  • Electrophoretic mobility shift assay (EMSA)
  • Gel shift assays
  • In vitro assays
  • Poly(A)-binding protein (PABP)
  • Polyacrylamide gel preparation
  • Protein-nucleic acid interactions
  • RNA and DNA binding

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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