Affinity assays using fluorescence anisotropy with capillary electrophoresis separation.
A novel approach to detecting affinity interactions that combines fluorescence anisotropy with capillary electrophoresis (FACE) was developed. In the method, sample is injected into a capillary filled with buffer that contains a fluorescent probe that possesses low fluorescence anisotropy. If proteins or other large molecules in the sample bind the fluorescent probe, their migration through the capillary can be detected as a positive anisotropy shift. Thus, the method provides both separation and confirmation of binding to the probe. Calculations based on combining the Perrin equation and dissociation constant were used to predict the effect of conditions on aniostropy detection. These calculations predict that low probe concentrations yield the best sensitivity while higher concentrations increase the dynamic range for detection of binding partner. The assay was applied to detection of G proteins using BODIPY FL GTPgammaS as the fluorescent probe. Experimental measurements exhibited trends in anisotropy with varying probe and protein concentrations that were consistent with the calculations. The limit of detection for G(alphai1) was 3 nM when the electrophoresis buffer contained 250 nM BODIPY FL GTPgammaS. FACE affinity assay is envisioned as a method that can quantify selected binding partners and screen complex samples for compounds that possess affinity for a particular small molecule that is used as a probe.
Whelan
Rebecca J
RJ
Department of Chemistry and Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109, USA.
Sunahara
Roger K
RK
Neubig
Richard R
RR
Kennedy
Robert T
RT
D019075:Electrophoresis, Capillary / Q000379:methods*; D005454:Fluorescence Polarization / Q000379:methods*; D005456:Fluorescent Dyes; D019204:GTP-Binding Proteins / Q000032:analysis; D049452:Green Fluorescent Proteins / Q000737:chemistry; D011485:Protein Binding; D011506:Proteins / Q000032:analysis; D013997:Time Factors
D005456:Fluorescent Dyes; D011506:Proteins; D049452:Green Fluorescent Proteins; D019204:GTP-Binding Proteins