Protocol to detect nucleotide-protein interaction in vitro using a non-radioactive competitive electrophoretic mobility shift assay

STAR Protoc. 2022 Dec 16;3(4):101730. doi: 10.1016/j.xpro.2022.101730. Epub 2022 Sep 30.

Abstract

Electrophoretic mobility shift assay (EMSA) is a classical and popular approach for DNA/RNA protein-binding affinity detection in vitro. This protocol describes a competitive EMSA assay using digoxigenin (DIG)-labeled probe, which solves the safety issues and limitations attributed to the short lifespan of the 32P-radiolabeled DNA probe. We detail steps for DNA probe preparation, protein-DNA mixture coincubation, EMSA, and competitive EMSA process. We optimize the standard DIG-ddUTP-labeling EMSA protocol to high sensitivity with reproducible results. For complete details on the use and execution of this protocol, please refer to Feng et al. (2022).

Keywords: Microbiology; Molecular biology; Molecular/Chemical probes; Protein biochemistry; Protein expression and purification.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Probes
  • DNA*
  • DNA-Binding Proteins* / metabolism
  • Electrophoretic Mobility Shift Assay
  • Nucleotides
  • RNA-Binding Proteins

Substances

  • DNA-Binding Proteins
  • DNA
  • DNA Probes
  • RNA-Binding Proteins
  • Nucleotides