A capillary electrophoresis mobility shift assay for protein-DNA binding affinities free in solution.

Abstract

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.