Generation of anti–glycated hCD59 polyclonal antibody. A: Glycated hCD59 peptide was synthesized with a glycated lysine in the K41 equivalent position (Kglu). B: Expression of recombinant hCD59 (rhCD59) in CHO cells was detected by immunoblot analysis with anti-FLAG antibody. Lanes 1 and 4, no expression of hCD59; lanes 2 and 3, expression of hCD59. C: Purified rhCD59 was incubated with and without glucose (0.5 mol/l at 37°C), and aliquots were tested at different time points
by immunoblotting with anti-hCD59 antibody (YTH53.1). Lane 1, rhCD59 (no glucose); lane 2, rhCD59 (glucose for 7 days); lane 3, rhCD59 (glucose for 21 days). D: Same samples as in C were immunoblotted with anti–glycated hCD59 antibody, and commercially available glycated albumin was used to control reactivity
with other glycated proteins. Lane 1, molecular marker; lane 2, rhCD59 (no glucose); lane 3, rhCD59 (glucose for 7 days); lane 4, rhCD59 (glucose for 21 days); lane 5, glycated albumin.