TY - CHAP
T1 - Characterization of cis-regulatory elements and transcription factor binding
T2 - Gel mobility shift assay
AU - Lin, Jim Jung Ching
AU - Grosskurth, Shaun E.
AU - Harlan, Shannon M.
AU - Gustafson-Wagner, Elisabeth A.
AU - Wang, Qin
PY - 2007/3/19
Y1 - 2007/3/19
N2 - 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.
AB - 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.
KW - A/T-rich region
KW - Antibody supershift assay
KW - Cardiac troponin T promoter
KW - Cardiac-specific trans-acting factor
KW - D module
KW - F module
KW - Gel mobility shift assay (GMSA)
KW - Gel retardation assay
KW - Gel-shift competition assay
KW - MEF2-like motif
KW - TCTG(G/C) direct repeat
UR - http://www.scopus.com/inward/record.url?scp=34447261176&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=34447261176&partnerID=8YFLogxK
U2 - 10.1385/1-59745-030-8:183
DO - 10.1385/1-59745-030-8:183
M3 - Chapter
C2 - 17568125
AN - SCOPUS:34447261176
SN - 1597450308
SN - 9781597450300
T3 - Methods in Molecular Biology
SP - 183
EP - 201
BT - Cardiac Gene Expression
A2 - Zhang, Jun
A2 - Rokosh, Gregg
ER -