Udp glo glycosyltransferase assay kit

The UDP-Glo assay was performed using a UDP-Glo Glycosyltransferase Assay kit (Promega) as described previously (22 (link)). Briefly, the GnT-V reaction was conducted in a 96-well white plate by incubating the purified GnT-V or its mutants with acceptor substrates for 2 h at room temperature in 10 μl of the reaction mixture that contained 10 mM ultrapure UDP-GlcNAc (supplied in the kit), an acceptor substrate, 125 mM MES (pH 6.25), 10 mM EDTA, 200 mM GlcNAc, 0.5% (v/v) Triton X-100, and 1 mg/ml BSA. The concentrations of the acceptor substrates were defined so that 100 pmol of N-glycans were added per well based on the number of N-glycans they have (α1AGP, 5; transferrin, 2; SOD3, 1; CCL1, 1; and SGP, 1). After the reaction, 15 μl of the GnT-V buffer (125 mM MES (pH 6.25), 10 mM EDTA, 0.5% (v/v) Triton X-100, and 1 mg/ml BSA) was added to each well followed by adding 25 μl of the UDP detection reagent (supplied in the kit). Then, the plate was incubated in the dark for 1 h at room temperature. Chemiluminescence signals were measured using a SYNERGY H1 microplate reader (BioTek). The duplicate measurements for each sample were performed, and the independent experiments were repeated at least three times for each acceptor substrate.

The effect of the CANT1 variant (Glu215Lys) on the nucleotidase activity of CANT1 towards uridine diphosphate (UDP) was examined, similar to previously described.20 (link) In brief, the assay mixture contained 5 µL of 10-times diluted conditioned media, 50 mM 2-(N-morpholino) ethanesulfonic acid-NaOH (pH 6.5), and 1 mM CaCl₂, with 0.2 mM UDP (Promega) as the substrate, in a total volume of 50 µL. The mixture was incubated at 37°C for 10 min, then inactivated by incubation at 95°C for 3 min. To determine the amount of unreacted UDP, the reaction product was mixed with the UDP detection reagent, containing an enzyme to convert UDP to ATP (UDP-Glo Glycosyltransferase Assay Kit, Promega). The resultant ATP was measured using a luciferase/luciferin reaction and the luminescent signals detected using a Victor X4 luminometer (PerkinElmer).

The UDP-Glo assay was performed using a UDP-Glo Glycosyltransferase Assay kit (Promega), as described previously (25 (link)). Briefly, the GnT-IV reaction was conducted in a 96-well white plate by incubating purified GnT-IVs with acceptor asialoagalacto substrates for 2 h at room temperature in 10 μl of the reaction mixture that contained 10 mM ultrapure UDP-GlcNAc (supplied in the kit), an acceptor substrate, 25 mM Mes (pH 7.7), 0.5% (v/v) Triton X-100, 5 mg/ml BSA, and 7.5 mM MnCl₂. The concentrations of the acceptor substrates were defined so that 100 pmol of N-glycans were added per well based on the number of N-glycans they have (GnGnbi-PA, 1; transferrin, 2; α1AGP, 5; fibrinogen, 6; haptoglobin, 4; α1-antitrypsin, 3; and IgG, 2). After the reaction, 15 μl of the GnT-IV buffer (25 mM Mes [pH 7.7], 0.5% [v/v] Triton X-100, 5 mg/ml BSA, and 7.5 mM MnCl₂) was added to each well followed by adding 25 μl of the UDP detection reagent (supplied in the kit). Then, the plate was incubated in the dark for 1 h at room temperature. Chemiluminescence signals were measured using a SYNERGY H1 microplate reader (BioTek). The duplicate measurements for each sample were performed.

UDP-rhamnose hydrolysis assay was carried out using the UDP-Glo Glycosyltransferase Assay kit (Promega) following the manual. Briefly, 0.4 μg (~0.5 μM) of AvrB and 8 μg of GST-RIN4 (~6 μM) were included in the reaction in the same buffer as in rhamnosylation assay above (final volume of 25 μl). UDP-rhamnose (20 μM) was included as donor. Reactions were performed at room temperature for 30 min. Equal volume of UDP detection reagent was then added and incubated for additional 60 min. A BMG Labtech CLARIOstar Plus Microplate Reader was used to record the luminescence signals. The mean values of triplicate readings for various groups of samples were analyzed and compared by ordinary one-way analysis of variance (ANOVA) and Tukey test (P < 0.05) using GraphPad Prism software.