Bead-Based Protein Binding Assay

The general methods for bead-based assays have been described (Nishikori et al., 2012 (link)). In the assay, streptavidin-coated Dynabeads M280 beads (Invitrogen) at 20 μg/ml were incubated with 5 nM biotinylated target protein diluted in BSS/EDTA/DTT buffer (50 mM Tris–HCl, 150 mM NaCl, pH 8, 1 mg/ml bovine serum albumin, 1 mM EDTA, 0.1 mM DTT) for 30 min. The remaining free biotin-binding sites of streptavidin on the M280 beads were blocked with 5 μM free biotin for 30 min. Ten microliters of the target-immobilized beads were transferred to the wells of a 96-well filter plate (MultiScreen HTS HV, 0.45 μm, Millipore), drained using a vacuum manifold (MultiScreen HTS Vacuum Manifold, Millipore), and washed with 100 μl of BSS/EDTA/DTT buffer. Next, a biotinylated protein (biotinylation of the proteins was checked by their ability to bind to streptavidin beads) to be tested at various concentrations was added to individual wells and incubated for 30 min with gentle shaking. Then the wells of the filter plate were washed twice with 150 μl of the buffer, 20 μl of 10 μg/ml SAV-Dylight650 (ThermoFisher) in the buffer was added to the wells, and the plate incubated with shaking for 30 min. The wells were washed again and the beads resuspended in 140 μl buffer and analyzed using a Guava EasyCyte 6/l flow cytometer (Millipore).