DNA-binding affinities of MyoD and E47 homo- and hetero-dimers by capillary electrophoresis mobility shift assay.
A simple capillary electrophoresis mobility shift assay (CEMSA), with no gel and uncoated capillaries, for the accurate determination of protein-DNA affinities free in solution was applied to constructs of the MyoD/E47 DNA-binding proteins. The determined affinities are compared to those obtained by EMSA. MyoD-E47 covalent heterodimer binds DNA more tightly (Kd=1.8 nM) than MyoD (Kd=14.2 nM) or E47 (Kd= 11.5 nM) covalent homodimers. The effect of non-specific DNA on binding affinities was more important than salt concentration in the MyoD/E47 series. Application of this method to the MyoD/E47 system demonstrates the generality of our CEMSA.
Foulds
G J
GJ
Department of Chemistry, University of Virginia, Charlottesville 22901, USA.
Etzkorn
F A
FA
D000465:Algorithms; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D019075:Electrophoresis, Capillary; D005782:Gels; D018257:Helix-Loop-Helix Motifs; D017570:MyoD Protein / Q000737:chemistry*; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors
D004268:DNA-Binding Proteins; D005782:Gels; D017570:MyoD Protein; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors; D004247:DNA