In this assay, recombinant full-length Hsp90a and TRAP1 containing C terminal His tags purified from SF9 cells were employed. ATP and MDCC-PBP were diluted to two times their final concentrations in the assay buffer and dispensed at 10 pL per well. The buffer consisted of 50 mM Tris, pH 7.5, 6 mM MgCl2, 20 mM KCl, and 1 mM freshly added DTT. Low volume plates were used to conserve MDCC-PBP, the most limited reagent. Full-length Hsp90a and TRAP1 were diluted to two times their final concentrations in assay buffer and 50 pL per well were dispensed into 96-well polypropylene plates. Compounds tested were diluted to 40 times their final concentrations in a separate 96-well polypropylene plate using 100% DMSO. Serial dilutions were made in 100% DMSO; 2.5 pL of 40 times diluted compounds or DMSO alone were added to the 50 pL of Hsp90a and TRAP1.
The proteins were first set up in this larger volume to accurately add compound and keep the final DMSO concentration at 2.5%. After mixing, 10 pL of the protein-compound-DMSO solutions were combined with the 10 pL of ATP-MDCC-PBP in the low volume plates. An initial reading was taken before the plates were sealed and placed in a 37 °C incubator. Background wells consisted of MDCC-PBP plus ATP only and no protein. Plates were read in a VICTOR2 equipped with a 405-nm excitation filter and a 460-nm emission filter. The increase in MDCC-PBP fluorescence at 460 nm was monitored every 30 to 60 min. The plates were unsealed before each reading and resealed before returning to 37°C.
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