Amplified Luminescent Proximity Homogeneous Assay: A Bead-Based Proximity Assay to Screen Small Molecules Inhibiting Protein-Protein Interactions

Add glutathione donor beads at 10 micrograms per milliliter concentration in PBS. Add GST-FKBP51 to a final concentration of 10 micrograms per milliliter, and incubate the reaction for 10 minutes at 25 degrees Celsius in the dark.

Make serial dilutions of the test compound in DMSO. Add 0.25 microliters of test compound dilutions in triplicate, in the corner of each well of a 384-well plate.

For negative control, add 0.25 microliters of DMSO, and for positive control, add 0.25 microliters of Hsp90 C-terminal peptide in the wells.

Add 22.5 microliters of the solution containing glutathione donor beads with GST-tagged proteins to each well. Shake the plate thoroughly with hands and incubate in the dark at 25 degrees Celsius for 15 minutes.

Dilute the acceptor beads with the attached Hsp90 C-terminal peptide to 100 micrograms per milliliter concentration in 0.5x PBS. Add 2.25 microliters of diluted acceptor beads to each well. Shake and incubate the plate in the dark for 15 minutes as demonstrated.

Turn on the plate reader instrument, measure the signal of the plate, and analyze the data as described in the text manuscript.