Characterization of cis-regulatory elements and transcription factor binding: gel mobility shift assay.
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.
Lin
Jim Jung-Ching
JJ
Department of Biological Sciences, University of Iowa, Iowa City, USA.
Grosskurth
Shaun E
SE
Harlan
Shannon M
SM
Gustafson-Wagner
Elisabeth A
EA
Wang
Qin
Q
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D005753:Gastric Mucosa / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051381:Rats; D050436:Regulatory Elements, Transcriptional / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000235:genetics*; D020107:Troponin T / Q000235:genetics
D014157:Transcription Factors; D020107:Troponin T