Gdp azido fucose

Before the CuAAC experiments, glycosyltransferase reactions were carried out with 1µg of WT POFUT1 or one mutated POFUT1 variant, mixed with 2 nmoles of GDP-azido-fucose (R&D Systems Inc., Minneapolis, MN, USA) as recommended by the manufacturer (R&D Systems) and 2 µg of purified EGF-LD in 25 µL of reaction buffer (25 mM Tris, 5 mM CaCl₂, 10 mM MnCl₂, pH 7.5), and then incubated for 1 h or 20 h at 37 °C.
For mass spectrometry analysis, 1 µg of WT or mutated POFUT1 was incubated with 2 µg of purified EGF-LD and 2 nmoles of GDP-fucose in 20 µl of reaction buffer and incubated for 20 h at 37 °C.

Before mass spectrometry analyses, 2.5 µg recPOFUT1 were incubated with 5 µg isolated EGF-LD and 0.1 mM GDP-fucose in 20 µl of reaction buffer (25 mM Tris, 5 mM CaCl₂, 10 mM MnCl₂, pH 7.5) and incubated overnight at 37 °C as previously described [37 (link)]. Additional experiments were carried out with 5 µg isolated EGF-LD III incubated with 2.5 µg of each of the recombinant enzymes (recPOFUT1, recLFNG) and 0.1 mM of each appropriate nucleotide sugar (GDP-fucose, UDP-GlcNAc) in 20 µL of reaction buffer as above.
Before click chemistry experiments, in vitro glycosyltransferase reactions were carried out with 1 µg recPOFUT1 mixed with 2 nmoles GDP-azido-fucose (R&D Systems Inc., Minneapolis, MN, USA) and 2 µg isolated EGF-LD or 1 µg recombinant WIF1 protein variant in 25 µL reaction buffer and incubated overnight at 37 °C. For O-fucose extension with GlcNAc, 1 µg recLFNG was mixed with 2 nmoles UDP-azido-GlcNAc (R&D Systems Inc., Minneapolis, MN, USA) and 1 µg recombinant WIF1 protein variant for overnight incubation at 37 °C before being subjected to click chemistry.