| Assay Method Information | |
| | Homogeneous Time Resolved Fluorescence (HTRF) Assay |
| Description: | An Homogeneous Time Resolved Fluorescence (HTRF) assay was performed in a manner similar to previous reports on HTRF protein protein assays as reviewed by Mathis (Clin Chem, 2005). The assay procedure was performed as follows: reactions were performed in 20 ul final volume in 384-well black low volume microtiter plates (Greiner). The final reaction buffer contained 29 mM phosphase buffer (pH 7), 10 mM HEPES buffer (pH 7.4), 68.5 mM NaCl, 1.4 mM KCl, 400 mM KF 0.05% (w/v) pluronic acid (P104, Sigma Aldrich) and 1% (v/v) DMSO. His6-tagged integrase (78 nM final concentration) was incubated with mannose binding protein fused to the Delta 325 carboxy terminal integrase binding domain of LEDGF in the presence of compound for 2 hours at room temperature. Both these protein regents were supplied by Prof. Zeger Debyser of Katholieke Universiteit Leuven, Leuven, Belgium. The compounds were added at varying concentrations spanning a wide range from 0.1 up to 100 uM. |
| Affinity data for this assay | |
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