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Assay Method Information |
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| | β-1,4-Galactosyltransferase I Assay |
| Description: | β4GalT activity was assayed using UDP-Gal as glycosyl donor and (6-esculetinyl) β-D-glucopyranoside (esculine) as glycosyl acceptor. Assays were performed in a total volume of 200 μL. The reaction mixtures contained reagents in the following final concentrations: 50 mM Hepes buffer (pH 5.4), 10 mM MnCl2, 2.0 mg/mL BSA, 200 μM esculine, 40 μM UDP-Gal, 10 μL MeOH and potential inhibitors 1-6 at 0.8 mM concentration. The enzymatic reactions were initiated by the addition of 1 mU β4GalT and incubated at 30 °C for 60 min. Inactivation was quickly done by placing of the reaction solutions for 3 min in a thermo block set to 90 °C. Thesolutions were diluted with water (300 μL) and centrifuged for 20 min, filtered through M.E. Cellulose filter (0.2 μm × 13 mm) and the filtrate was injected into RP-HPLC system. |
| Affinity data for this assay | |
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