Characterization of cis-regulatory elements and transcription factor binding: Gel mobility shift assay

Jim Jung Ching Lin, Shaun E. Grosskurth, Shannon M. Harlan, Elisabeth A. Gustafson-Wagner, Qin Wang

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

To understand how cardiac gene expression is regulated, the identification and characterization of cfr-regulatory elements and their trans-acting factors by gel mobility shift assay (GMSA) or gel retardation assay are essential and common steps. In addition to providing a general protocol for GMSA, this chapter describes some applications of this assay to characterize cardiac-specific and ubiquitous frarw-acting factors bound to regulatory elements [novel TCTG(G/C) direct repeat and A/T-rich region] of the rat cardiac troponin T promoter. In GMSA, the specificity of the binding of trans-acting factor to labeled DNA probe should be verified by the addition of unlabeled probe in the reaction mixture. The migratory property of DNA-protein complexes formed by protein extracts prepared from different tissues can be compared to determine the tissue specificity of trans-acting factors. GMSA, coupled with specific antibody to DNA-acting factor (antibody supershift assay), is used to identify proteins present in the DNA-protein complex. The gel-shift competition assay with an unlabeled probe containing a slightly different sequence is a powerful technique used to assess the sequence specificity and relative binding affinity of a DNA-protein interaction. GMSA with SDS-PAGE fractionated proteins allows for the determination of the apparent, molecular mass of bound trans-acting factor.

Original languageEnglish (US)
Title of host publicationCardiac Gene Expression
Subtitle of host publicationMethods and Protocols
EditorsJun Zhang, Gregg Rokosh
Pages183-201
Number of pages19
DOIs
StatePublished - Mar 19 2007
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume366
ISSN (Print)1064-3745

Keywords

  • A/T-rich region
  • Antibody supershift assay
  • Cardiac troponin T promoter
  • Cardiac-specific trans-acting factor
  • D module
  • F module
  • Gel mobility shift assay (GMSA)
  • Gel retardation assay
  • Gel-shift competition assay
  • MEF2-like motif
  • TCTG(G/C) direct repeat

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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