| Solution Information | help | |
| Enzyme: | Large T antigen | |
| inhibitor: | BDBM32020 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Primary Assay Conditions: The assay was conducted in 1536-well black, clear-bottom microplates. Assay buffer was 100mM Tris-HCl, 20mM KCl, 6mM MgCl2, and 0.1mg/mL BSA, pH 7.4. Initially, 250nl of test compound (20uM final concentration) in 4% DMSO/assay buffer was added to the respective plate wells. Total reaction volume (without endpoint reagent) was 1.25ul. Total read volume (with endpoint reagent) was 5 ul. Enzyme and buffer volumeswere dispensed using a BioRAPTR FRD (Beckman Coulter, Fullerton, CA). Compounds were dispensed using an Echo 500 Series Liquid Handler (Labcyte, Sunnyvale, CA). Purified TAg was diluted to a concentration of 0.0144 ug/ul in assay buffer. 750nl of TAg (14.4ng/ul, 10.8ng total) in assay buffer was added to each well and allowed to incubate for 30m at ambient temperature. Following incubation, 250nl of 5mM ATP (A7699, Sigma-Aldrich, St. Louis, MO) in assay buffer was added to the respective wells, and incubated for 120m at ambient temperature. Final DMSO c | |
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