SPR binding experiments were performed at 25 °C on a Biacore T200 instrument using a CM-5 chip (GE Healthcare) with coupled anti-GST antibody as described previously48 (link)
. GST-tagged UBE2K variants were coupled on the chip and a serial dilution of Ub in running buffer containing 25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM DTT and 0.005% (v/v) Tween-20 was used as analyte. Two technical replicates were performed and data were analyzed with BIAevalution (GE Healthcare) and fit to a one-site binding model using Prism (GraphPad).
. GST-tagged UBE2K variants were coupled on the chip and a serial dilution of Ub in running buffer containing 25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM DTT and 0.005% (v/v) Tween-20 was used as analyte. Two technical replicates were performed and data were analyzed with BIAevalution (GE Healthcare) and fit to a one-site binding model using Prism (GraphPad).
