Udp glotm glycosyltransferase assay kit

Recombinant SseK1, SseK2 and point mutant proteins were prepared as described and GT kinetics were measured using UDP-Glo^TM Glycosyltransferase Assay kit (Promega, #V6961) by manufacturer’s instruction. Enzyme reaction buffer (ERB) was prepared as 25 mM Tris-HCl [pH 7.5], 50 mM NaCl, 4 mM MnCl₂, 1 mM DTT and reaction was eliminated by using the nucleotide detection buffer. For preparing the acceptor-substrate, L-arginine (Duchefa BIOCHEMIE, > 98.5% purity) was dissolved in ERB. Synthetic peptides of GAPDH (Genscript, > 90% purify) was purchased and dissolved in ERB. White 96-well plates (ThermoScientific) were used for luminescence assay and the plate was read by using luminometer (VictorX5, PerkinElmer). Kinetics parameters were calculated using GraphPad Prism5 ver.5.03 software. The points represent an average of two samples and error bars represent mean ± S.D. The final specific activity of the transfer reaction was corrected considering the hydrolysis reaction, which was performed using SseK1/SseK2, UDP-GlcNAc, and MnCl₂.

The UDP-GloTM Glycosyltransferase Assay Kit was used as specified by the manufacturer (Promega). OGT (200 nM) in 25 mM Tris-HCL buffer pH 7.5, 12.5 mM MgCl₂, 0.06 mg/mL BSA, 1 mM DTT, 50 µM OGT peptide substrate, and 100 µM UDP-GlcNAc were used in the reactions, which also included avasimibe at concentrations ranging from 6 to 200 µM. The reactions were incubated at 22 °C for 1 h, and the luminescence signals were measured by using a FLUO star microplate reader (BMG Labtech, Cary, NC, USA).