Fluorescence polarization detection for affinity capillary electrophoresis
Affinity capillary electrophoresis (ACE) with laser-induced fluorescence polarization (LIFP) detection is described, with examples of affinity interaction studies. Because fluorescence polarization is sensitive to changes in the rotational motion arising from molecular association or dissociation, ACE-LIFP is capable of providing information on the formation of affinity complexes prior to or during CE separation. Unbound, small fluorescent probes generally have little fluorescence polarization because of rapid rotation of the molecule in solution. When the small fluorescent probe is bound to a larger affinity agent, such as an antibody, the fluorescence polarization (and anisotropy) increases due to slower motion of the much larger complex molecule in the solution. Fluorescence polarization results are obtained by simultaneously measuring fluorescence intensities of vertical and horizontal polarization planes. Applications of CE-LIFP to both strong and weak binding systems are discussed with antibody-antigen and DNA-protein binding as examples. For strong affinity binding, such as between cyclosporine and its antibody, complexes are formed prior to CE-LIFP analysis. For weaker binding, such as between single-stranded DNA and its binding protein, the single-stranded DNA binding protein is added to the CE separation buffer to enhance dynamic formation of affinity complexes. Both fluorescence polarization (and anisotropy) and mobility shift results are complementary and are useful for immunoassays and binding studies.