UDP-GloTM Glycosyltransferase Assay

Detection of free UDP after hydrolysis of the sugar nucleotide was performed using the UDP-Glo^TM Glycosyltransferase Assay Kit (Promega Corporation), which detects UDP after UDP-sugar hydrolysis or transfer by converting UDP to light (measured in Relative Luminescence Units) in a luciferase type reaction. A standard curve using 0–25 µM UDP was performed, and the range of measurements was determined to be in the linear range of detection (in all cases, UDP-sugar consumption was <10% of the starting amount), where the luminescence detected is directly proportional to UDP concentration. Following the manufacturer's protocol, each sugar-nucleotide hydrolysis reaction was combined in a ratio of 1:1 (5 µL:5 µL) with the UDP-Glo^TM Detection Reagent in independent wells of a white, flat bottom 384-well assay plate (Corning) and allowed to incubate at ambient temperature. After 1 h of incubation, luminescence was measured in triplicate using a GloMax^®-Multi+ Microplate Luminometer (Promega Corporation).