| Assay Method Information | |
| | Inhibition Assay |
| Description: | Test compound is dissolved in DMSO 500× the highest final concentration desired in the IC50 assay. 30 ml of deionized water is pre-warmed to 37° C. and all kit components are placed on ice. For each well of column 1, 149.4 μL of NADPH-Cofactor Mix, (187.5 μl of Cofactors, 150 μl of G6PDH, 100 μl of Control Protein and 14.56 ml of 37° C. water) are added. In each well from Column 2 to 12, 100 μl of Cofactor/DMSO mix (40 μL DMSO in 9.96 ml of NADPH-Cofactor Mix) are added. To each well of column 1, 0.6 μl of test compound or positive control are added. 50 μl from each well of column 1 are serially diluted to column 8. The extra 50 μl from column 8 are discarded. The plate is covered and pre-incubated at 37° C. for 10 minutes. Preparation of enzyme/substrate mix: 7.92 ml of pre-warmed deionized water, 75 μl of enzyme, 3 μl of 10 mM AMMC and 2 ml of pre-warmed buffer are mixed. After the pre-incubation time (10'), 100 μl of enzyme/substrate mix to each well from column 1 to 10 are added. The plate is incubated at 37° C. for 30 minutes. After this time, 75 μl of Stop Reagent to each well are added. For blank controls, 100 μl of enzyme/substrate mix are added to columns 11 and 12. |
| Affinity data for this assay | |
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