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| | GAR Transformylase Inhibition Assay |
| Description: | GARFTase activity was determined by measuring the rate of 5,8-dideazatetrahydrofolate production in the presence of varying concentrations of antifolate inhibitors using an established spectrophotometric protocol.39 A 150 μL total volume containing 30 μM α,β-GAR, 5.4 μM 10-CHODDF and antifolate (stock solution dissolved in DMSO) in 0.1 M HEPES (pH 7.5) was preincubated at 37 °C in a UV. To initiate the assay, 150 μL of 20 nM His-GARFTase or buffer was added, and the plate was shaken for 5 s. Absorbance changes at 295 nm wererecorded at 15 s intervals over 20 min using a Synergy H1M plate reader (BioTek). For each antifolate, triplicate assays were performed at 20 different drug concentrations. |
| Affinity data for this assay | |
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