| Assay Method Information | |
| | ELISA-based PBD-binding inhibition assay |
| Description: | An ELISA-based PBD-binding inhibition assay was performed essentially as described previously. Briefly, a biotinylated PBIP1 p-T78 peptide (i.e., Biotin-Ahx-C-ETFDPPLHSpTAI-NH2) was first diluted with 1× coating solution (SeraCare, Gaithersburg, MD) to the final concentration of 0.3 μM, and then 100 μl of the resulting solution was immobilized onto a 96-well streptavidin-coated plate (Nalgene Nunc, Rochester, NY). The wells were washed once with PBS+0.05% Tween20 (PBST) and incubated with 200 μl of PBS plus 1% BSA (blocking buffer) for 1 h to prevent non-specific binding. HEK293A lysates expressing HA-EGFP-Plk1 were prepared in a TBSN [20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% NP-40, 5 mM EGTA, 2 mM MgCl2, 1.5 mM EDTA, and protease inhibitor cocktail (Roche)]+20% DMSO buffer ( 20 μg total lysates in 100 μl buffer), mixed with the indicated amount of the competitors (p-T78 peptide and its derivative compounds) for 30 min, provided onto the biotinylated peptide-coated ELISA wells, and then incubated with constant rocking for 1 h at 25° C. Following the incubation, ELISA plates were washed 5 times with PBST. To detect bound HA-EGFP-Plk1, the plates were probed for 2 h with 100 μl/well of anti-HA antibody at a concentration of 0.5 μg/ml in the blocking buffer and then washed 5 times. The plates were further probed for 1 h with 100 μl/well of HRP-conjugated secondary antibody (GE Healthcare) at a 1:1,000 dilution in the blocking buffer. The plates were washed 5 times with PBST and incubated with 100 μl/well of 3,3′,5,5′-tetramethylbenzidine solution (Sigma-Aldrich, St. Louis, MO) as substrate until a desired absorbance was reached. The reactions were stopped by the addition of 100 μl/well of stop solution (Cell Signaling Technology, Danvers, MA). The optical density (O.D.) was measured at 450 nm by using a Perkin-Elmer Enspire Multimode Plate reader (PerkinElmer, Inc., Boston, MA). Data obtained from more than three independent experiments were analyzed by GraphPad (San Diego, CA) Prism software version 7.[0568]Microsomal metabolic stability assays were performed as reported previously (Urban et al., Sci. Rep. 2017, 7 (1), 12758). PAMPA permeability was measured using a high throughput protocol, as reported (Sun et al., Bioorg. Med. Chem. 2017, 25 (3), 1266-1276). Aqueous solubility was determined by a published procedure (Sun et al., Bioorg. Med. Chem. 2019, 27 (14), 3110-3114). |
| Affinity data for this assay | |
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