Protein Kinase Interaction Screening

AlphaScreen assays were performed as described previously [23 (link)]. All recombinant proteins used here was synthesized using a wheat germ based cell-free 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 GST-Gag or GST-DHFR in 10 μl of kinase assay buffer (100 mM Tris–HCl pH8.0, 10 mM MgCl₂, 0.1% Tween20, 0.1% BSA) at 37°C for 1 h in one well of a 384-well Optiplate (Perkin Elmer, Foster City, CA. In accordance with the AlphaScreen IgG (protein A) detection kit (Perkin Elmer) instruction manual, 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 from the mixture were detected using an EnVision device (PerkinElmer) with the AlphaScreen signal detection program.