A capillary electrophoresis mobility shift assay for protein-DNA binding affinities free in solution.
Quantitative determination of dissociation constants for DNA-protein complexes will help clarify the molecular mechanisms of transcription, replication and DNA repair. A practical capillary electrophoresis mobility shift assay (CEMSA) for protein-DNA affinities free in solution is presented. The method is fast and simple, precise and general. The speed (<2 min separations) and simplicity derive from the use of an uncoated capillary with no gel matrix. The dissociation constant for GCNK58, a DNA-binding-region construct of the yeast transcription factor GCN4, binding to the AP1 DNA site was measured ( K d = 35 +/- 4 nM) to demonstrate the utility of the method.
Department of Chemistry, University of Virginia, McCormick Road, Charlottesville, VA 22901, USA.
G J
GJ
Foulds
F A
FA
Etzkorn
D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011494:Protein Kinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D012996:Solutions; D018808:Transcription Factor AP-1 / Q000737:chemistry / Q000378:metabolism*
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; D012996:Solutions; D018808:Transcription Factor AP-1; D004247:DNA; D011494:Protein Kinases