| Assay Method Information | |
| | HPLC-based Assay |
| Description: | Reactions were performed in duplicate at 22 C in 50 mM NaH2PO4 buffer (pH 7) with 20 nM enzyme and 20 mM fresh sodium dithionite. Small molecules were added from DMSO stocks to a final concentration of 1% DMSO. Concentration of UDP-Galf was held constant at the Km of the UGM homolog being analyzed, except for generation of Lineweaver-Burk plots. Reaction time and substrate concentration were varied for Lineweaver-Burk analysis, and each condition was tested in triplicate. Quenched reactions were analyzed using a CarboPac PA-100 column (Thermo Scientific) on an Agilent 1260 Infinity HPLC using an isocratic elution of 200 mM ammonium acetate (pH 8) to separate substrate and product peaks. |
| Affinity data for this assay | |
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