Expanding the capabilities of capillary electrophoresis for determining protein-DNA binding affinities: physiological conditions and mPEG-silane coating for sticky proteins

Measuring binding affinities of proteins to their specific sites in double-stranded (ds) DNA yields one of the fundamental parameters for characterizing protein-DNA interactions. Although many methods for determining binding affinities exist, all methods have limitations. Adding new approaches will enhance cross-validations and expand the range of targets that can be studied. We present inexpensive capillary electrophoresis (CE) methods for determining protein binding affinities to double-stranded DNA (dsDNA) in buffers with physiologically relevant salt concentrations, including divalent ions. We demonstrate the methods by monitoring the binding of the mismatch repair proteins Thermus aquaticus (Ta) MutS and Saccharomyces cerevisiae (Sc) MutSα to dsDNA containing a mismatch. We separate dsDNA and TaMutS-dsDNA complexes using bare fused silica (BFS) capillaries with pressure assistance. ScMutSα, like many proteins, sticks to BFS capillaries. Consequently, we developed a simple automated procedure to covalently coat BFS capillaries with commercially available methoxy polyethylene glycol silane (mPEG-silane), which minimizes protein sticking to capillary walls and allows separation of protein-dsDNA complexes using reverse polarity. The mPEG-silane coating remained sufficiently intact for over 24 h of continuous sample separation and was successfully regenerated. We show that the binding affinities of TaMutS and ScMutSα to dsDNA substrates determined by CE agree with those determined by fluorescence anisotropy or microscale thermophoresis.