Recombinant human st6gal1

Glycan array screening was performed by the Consortium for Functional Glycomics as previously published [10 (link)]. Glycans were desialylated using pan-specific neuraminidase in acetate buffer, pH 5.5 for 2 hours. Recombinant human ST6Gal1 (R&D Systems) was incubated on the desialylated chip for 3 hours at 37°C with or without 100 μM CMP-SA (Calbiochem) in ST6Gal1 Reaction Buffer. α2,6 sialic acids were detected with SNA-biotin (1 μg/ml; Vector) followed by sAv-Cy5 (0.5 μg/ml; JacksonImmuno) for detection.

IgG was purified from cell culture media or plasma samples by separation over a HiTrap Protein A HP Antibody Purification Column (GE Life Sciences) fitted to a GE Life Sciences Akta Purifier 10 HPLC. Binding and washing were done with 10 mM Tris, pH 7.5, and 150 mM NaCl. Elution of IgG was done with 50 mM Citrate, pH 4.5, and 150 mM NaCl. Purified IgG was buffered-exchanged into PBS (ELISA and long-term storage) or water (for HPLC glycan analyses), and purity validated by SDS-PAGE.
IgG was sialylated in vitro using recombinant human ST6Gal1 (R&D Systems). 50 μg of purified IgG was incubated in ST6Gal1 Reaction Buffer (50 mM Na Cacodylate, 5 mM MgCl₂, 0.5% Tx-100, 1 mg/ml BSA, pH 7) with 500 ng of ST6Gal1 and 100 μM CMP-SA (Neu5Ac) overnight (16 hours) at 37°C.