Effect of VWF N-linked glycans of free thiol content. (A) VWF at 0.5 μg/mL was incubated with 100μM MPB before quenching with 200μM GSH. Samples were captured onto microtitre plates with polyclonal anti-VWF antibodies. Bound VWF was detected with anti–VWF-HRP and bound MPB with streptavidin-HRP. (B) The free thiol content of the recombinant VWFpp was determined in the same way, except labeled VWFpp was captured with anti-VWFpp antibody. (C) Wild-type VWF was treated with PNGase F overnight at 37°C and then labeled with MPB and captured to microtitre plates with polyclonal anti-VWF antibody. Error bars represent mean ± SEM of 3 separate experiments each performed in triplicate.