High-Throughput AlphaScreen Assay Optimization

The AlphaScreen® assay (Perkin Elmer) was generally performed as previously described.^{55 (link)} In brief, compound plates (1 μL at 10 mM highest concentration; 3-fold, 10-point dilutions in DMSO) were diluted in 1× assay buffer (20 mM TRIS pH 8.0, 25 mM NaCl, 2 mM DTT and 0.05% Tween-20) to 1 mM using a Multimek robotic pipettor (Nanoscreen) and 1 μL was spotted into the wells of 384-well low-volume Proxiplates (Perkin Elmer). To these plates 9 μL of protein-peptide mix in 1× assay buffer was added by Multidrop (Thermo) to bring the final compound concentration to 100 μM and incubated for 30 min at room temperature. Next, 2 μL of a 1:1 mixture of streptavidin-conjugate donor and nickel-chelate or α-GST acceptor beads (45 μg/mL in 1× assay buffer) were added and the plates were allowed to incubate for an additional 30 min in the dark at room temperature. After incubation, the plates were read on an EnVision multi-label reader equipped with an HTS AlphaScreen laser (Perkin Elmer). The expression and purification of the constructs used in this assay was described previously^{56 (link)}.