DNA-PROTEIN BINDING ASSAYS FROM A SINGLE SEA-URCHIN EGG - A HIGH-SENSITIVITY CAPILLARY ELECTROPHORESIS METHOD

A capillary electrophoresis method has been developed to study DNA-pro tein complexes by mobility-shift assay, This method is at least 100 ti mes more sensitive than conventional gel mobility-shift procedures, Ke y features of the technique include the use of a neutral coated capill ary, a small amount of linear polymer in the separation medium, and us e of covalently dye-labeled DNA probes that can be detected with a com mercially available laser-induced fluorescence monitor, The capillary method provides quantitative data in runs requiring <20 min, from whic h dissociation constants are readily determined, As a test case we stu died interactions of a developmentally important sea urchin embryo tra nscription factor, SpP3A2: As little as 2-10 x 10(6) molecules of spec ific SpP3A2-oligonucleotide complex were reproducibly detected, using recombinant SpP3A2, crude nuclear extract, egg lysates, and even a sin gle sea urchin egg lysed within the capillary column.