AlphaScreen Assay for Protein-Protein Interactions

AlphaScreen assays were performed as described previously (Tadokoro et al., 2010 (link)). All recombinant proteins were synthesized using the wheat germ CFPS system, as described above. For each protein kinase, 1 μl of crude recombinant biotinylated construct from the human kinase library was incubated with 1 μl of crude FLAG-Vpx or FLAG-DHFR in 10 μl of kinase assay buffer (100 mM Tris-HCl [pH 8.0], 10 mM MgCl2, 0.1% Tween-20, 0.1% BSA) at 37°C for 1 h in one well of a 384-well OptiPlate (PerkinElmer, Foster City, CA, USA). Using the AlphaScreen IgG (protein A) detection kit (PerkinElmer), 15 μl of detection mixture containing 100 mM Tris-HCl [pH 8.0], 0.01% Tween-20, 1 mg/ml BSA, 5 μg/ml anti-FLAG antibody (GE Healthcare, Buckinghamshire, UK), 5 ng streptavidin-coated donor beads, and 5 ng anti-IgG (protein A) acceptor beads were added to each well, followed by incubation at 26°C for 1 h. AlphaScreen signals were detected on an EnVision device (PerkinElmer) using the AlphaScreen signal detection program.