Monoclonal ACPA from the clones 109 and C7 and anti-TNP antibodies were generated as described elsewhere30 (link)39 (link). For galactosylation, 1 mg of IgG was incubated with 10 μM UDP-galactose (Calbiochem) and 2,5 mU of β1-4 galactosyltransferase (Sigma) in 50 mM MOPS, pH7.2 with 20 mM MnCl2 for 48 h at 37 °C. For sialylation, 1 mg of IgG was incubated with 10 μM CMP-sialic acid (Calbiochem) and 10 mU of α2-6 sialyltransferase (Sigma) in 50 mM MES, pH 6,0 with 10 mM MnCl2 for 48 h at 37 °C. The reactions were confirmed with a lectin blot.
Harre U., Lang S.C., Pfeifle R., Rombouts Y., Frühbeißer S., Amara K., Bang H., Lux A., Koeleman C.A., Baum W., Dietel K., Gröhn F., Malmström V., Klareskog L., Krönke G., Kocijan R., Nimmerjahn F., Toes R.E., Herrmann M., Scherer H.U, & Schett G. (2015). Glycosylation of immunoglobulin G determines osteoclast differentiation and bone loss. Nature Communications, 6, 6651.
Other organizations :
Friedrich-Alexander-Universität Erlangen-Nürnberg, Leiden University Medical Center, Karolinska University Hospital, Karolinska Institutet
Incubation with 10 μM UDP-galactose and 2.5 mU of β1-4 galactosyltransferase
Incubation with 10 μM CMP-sialic acid and 10 mU of α2-6 sialyltransferase
dependent variables
Galactosylation of IgG
Sialylation of IgG
control variables
1 mg of IgG
50 mM MOPS, pH 7.2 with 20 mM MnCl2
50 mM MES, pH 6.0 with 10 mM MnCl2
Incubation time of 48 h at 37 °C
controls
Lectin blot to confirm the reactions
Annotations
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