Figure 5. Substituted cysteine labelling of WcaJ_Cysless derivatives containing cysteine replacements.

Whole bacteria were treated with thiol-blocking reagents MTSES and NEM prior to lysis and labelling with PEG-Mal. The figure is a composite of multiple experiments each involving three experimental samples plus untreated control and the molecular mass markers. All samples were separated on a 14% polyacrylamide gel and detected by 1:10,000 dilution of anti-FLAG M2 monoclonal antibody. The topology of the cysteine residue was determined by the ability of MTSES and NEM to prevent PEG-Mal labelling. PEG-Mal labelling is indicated by a band shift. * and ** indicate the position of WcaJ and PEGylated WcaJ, respectively. U, untreated control.