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

Academic Article

Abstract

  • To understand how cardiac gene expression is regulated, the identification and characterization of cis-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 trans-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 trans-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.
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    Keywords

  • Animals, Base Sequence, Cells, Cultured, Electrophoresis, Polyacrylamide Gel, Electrophoretic Mobility Shift Assay, Gastric Mucosa, Liver, Molecular Sequence Data, Muscle, Skeletal, Myocardium, Promoter Regions, Genetic, Protein Binding, Rats, Regulatory Elements, Transcriptional, Sequence Homology, Nucleic Acid, Transcription Factors, Troponin T
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    Author List

  • Lin JJ-C; Grosskurth SE; Harlan SM; Gustafson-Wagner EA; Wang Q
  • Start Page

  • 183
  • End Page

  • 201
  • Volume

  • 366