Flow chambers were prepared on mPEG passivated quartz slides doped with biotin PEG15 (link). Biotinylated antibodies were immobilized by incubating ~10 nM of antibody for 10 min on NeutrAvidin (Thermo) coated flow chambers. Prism type total internal reflection fluorescence (TIRF) microscope was used to acquire the single molecule data40 (link). Samples with fluorescent protein tag were serially diluted to obtain well-isolated spots on the surface upon 20 min of incubation over immobilized antibody surface. All dilutions were made immediately before addition to the flow chamber in 10 mM Tris-HCl pH 8.0, 50 mM NaCl buffer with 0.1 mg/ml bovine serum albumin (New England Biolabs), unless specified. Unbound antibodies and sample were removed from the channel by washing with buffer twice between successive additions. For immunofluorescence detection, immunoprecipitated complexes were incubated with a different antibody against prey protein (~10 nM) for 20 min and fluorescent-dye-labeled secondary antibody (2–5 nM) for 5 min before imaging. Single molecule analysis was performed using scripts written in Matlab.