Dj. Lecaptain et al., Characterization of DNA-protein complexes by capillary electrophoresis-single molecule fluorescence correlation spectroscopy, ANALYST, 126(8), 2001, pp. 1279-1284
A high-speed capillary electrophoresis mobility shift assay (CEMSA) for det
ermining the binding ratios of DNA-protein complexes in solution is demonst
rated. Single molecule fluorescence correlation spectroscopy (FCS) was used
to resolve the bound and unbound fluorescently labeled DNA molecules as th
ey flowed continuously through a fused silica capillary under the influence
of an applied electric field. Resolution of the bound and unbound complexe
s was based on the difference in their electrophoretic mobilities, and was
accomplished without the need to perform a chemical separation. Data suffic
ient to perform the analysis was acquired in less than 10 s, compared to th
e minutes that are normally needed to carry out such measurement via CE sep
aration, The binding ratios were determined with 5 to 10% precision and agr
eed with the results obtained by CE separation within experimental error. T
he resolution of the CEMSA based FCS analysis (CEMSA-FCS) was significantly
higher than for the analysis performed by conventional diffusional FCS, du
e to the higher mass sensitivity of the electrophoretic mobility compared t
o the translational diffusion coefficient. Fluorescently labeled 39-mer sin
gle stranded DNA (ssDNA) and the single stranded binding protein (SSB) from
Escherichia coli was used as the model system. The dissociation constant o
f the ssDNA-SSB complex was estimated to be approximate to 2 nM based on th
e CEMSA-FCS analysis.