| Assay Method Information | |
| | In Vitro Evaluation of Bromodomain Inhibitors by Homogeneous Time Resolved Fluorescence |
| Description: | HTRF reagents and buffers were purchased from Cisbio Bioassays. The assay used a terbium (Ill) cryptate donor reagent conjugated to an anti-GST antibody (MAb anti-GST-Tb; GSTTLA), a streptavidin-conjugated acceptor reagent (streptavidin-d2) and Cisbio proprietary buffers (EPlgeneous Binding Domain Diluent and Detection buffer, respectively). GST-tagged bromodomains (BDs) were expressed in E. coli and purified using standard procedures. Incubation of GST-tagged BDs with biotinylated acetylated H4 peptide (H4K5acK8acK12acK16ac, here named H4ac4) brings the donor and acceptor into close proximity and allows for a FRET reaction. GST-tagged proteins in 25 mM Hepes pH 7.5, 150 mM NaCl, 0.5 mM DTT were assayed at a final concentration of 5 nM. Biotinylated H4ac4 peptides were used at a final concentration of 50, 600 nM in assays involving Brd4 BD1, Brd4 BD2, respectively. The antibody-conjugated donor was used at 0.5 nM and the streptavidin-conjugated acceptor was used at ⅛ of the H4ac4 peptide concentration. Inhibitors were tested by performing an eleven-point dilution series with a maximal final concentration of 20 mM. These concentrations allowed a fixed DMSO concentration at 0.2%, critical for a Z′ factor ≥0.8. Components were incubated at 4° C. for 4 h (BD1) or for 24 h (BD2). Experiments were performed in 384-well white plates (Greiner ref. 781080) and analyzed in a ClarioStar plate reader (BMG LABTECH, excitation at 330 nm and emission at 620 and 665 nm, corresponding to the donor and acceptor emission peaks, respectively; the 665/620 ratio is used to calculate the specific HTRF signal) with an integration delay of 60 μs and an integration time of 400 μs. |
| Affinity data for this assay | |
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