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| Solution Information | help | |
| Enzyme: | PC4 and SFRS1-interacting protein | |
| inhibitor: | BDBM4293 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Assay Overview: The purpose of this assay is to determine dose response curves of compounds identified as activators of the premature multimerization of IN in a set of previous experiments entitled: "Counterscreen for inhibitors of LEDGF/p75-dependent integration: TR-FRET-based biochemical high throughput counterscreen assay to identify activators of HIV-1 Integrase multimerization" (AID 3053131). This assay monitors the interaction between two full length wild type HIV-1 IN proteins: one containing an N-terminal hexa-histidine tag (6xHis) and the other a C-terminal FLAG tag. Anti-6xHis-XL665 and anti-FLAG-EuCryptate antibodies allow fluorescence energy transfer upon IN-IN interaction (4). In this assay, test compounds are incubated with IN mix (20nM 6xHis-In and 20nM FLAG-IN final concentration) in assay buffer 20 mM Hepes, pH 7.5, buffer containing 1 mM DTT, 10 mM MgCl2, 10% glycerol, 0.05% Brij-35, 0.1 mg/ml BSA). After 3 hr incubation time, Antibody mix is added to plates (1.25 | |
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