Fig. 4. The determination of affinities of anti-Rab4 and anti-Rab5 to GST-Rab fusion proteins by surface plasmon resonance. Surface plasmon resonance technology was used to measure the binding of anti-Rab4 monoclonal antibody to a cuvette coated with GST-Rab4 (A), and the binding of anti-Rab5 monoclonal antibody to a GST-Rab5-coated cuvette (B). Antibody was added at the indicated concentrations, binding was monitored for 10 minutes, cuvettes were acid washed to remove antibody, and antibody was re-added. An equilibrium plot of final signal versus antibody concentration (C) was used to fit a curve for the apparent affinities of the antibodies, yielding K_d values of 2 and 22 nM for anti-Rab4 and anti-Rab5 monoclonal antibodies, respectively. (D) Endocytic vesicles (total protein 60 µg) were incubated with 0.5 µM GST-Rab4 for 15 minutes at 37°C. Following extensive washing vesicles were immunoblotted with monoclonal antibody against Rab4.