| Solution Information | help | |
| Enzyme: | Phosphotransferase | |
| inhibitor: | BDBM61971 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Assay protocol used as developed for the original High Throughput Screening campaign The basic procedure for the TbHK1 HTS assay follows a stepwise addition of reaction mixture components as follows: 1 15 uL of a 30 uM concentration of test compound is added to appropriate wells (final compound concentration 10 uM). 2 15 uL of a glucose + ATP + MgCl2 + NAD+ + G6PDH mixture is added with final concentrations of 0.5mM, 0.35mM, 1.5mM, 3mM, and 0.006mUnits/uL, respectively. 3 15 uL of TbHK1 enzyme is added per well (final 0.5ng/uL). 4 Reaction incubates for 2 hours at room temperature. 5 5 uL EDTA is added (final 50mM). 6 Data was captured at A340 and represents the increase in NADH in the reaction mixture. | |
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