| Solution Information | help | |
| Enzyme: | Heat shock protein HSP 90-alpha | |
| inhibitor: | BDBM32170 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Compounds identified as active from the primary screen, AID 429 - HTS for Tumor Hsp90 Inhibitors, were selected for testing in this assay. Each compound was tested in 6 concentration, 1:2 serial dilutions starting at various starting concentrations. Each concentration was tested in triplicate. Reagents: 1. SKBr3 cells growing in RPMI-1640 medium with 10% FBS and 1% P/S. 2. TBST 3. Ice-cold TBST 4. −20 deg C Methanol 5. SuperBlock 6. Primary antibodies (anti-Hers) and secondary antibodies (anti-rabbit HRP-linked antibody ) in SuperBlock. 7. Chemiluminescent substrate solution Procedure: 1. Seed SKBr3 cells, 7X103 /well in 384-well plates, 50 ul medium/well 2. Incubate cells overnight, add 0.5 ul of compound (1 compound / well) 3. Incubate the cells with comppound for 24 hr. 4. Wash cells twice with ice-cold TBST (70#l) . 5. Methanol (70 #l at −20 deg C) was added to each well, and the plate was left at 4 deg C for 10 min. 6. Methanol was removed by washing with T | |
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