Figure 4. Detection of ¹⁴C-galactosylated endogenous acceptor co-migrating with WciS–His₆.

Recombinant proteins (12K and 50K supernatants) were prepared from E. coli BL21 (DE3) expressing WciS–His₆ (lanes 1–6, 10 and 11) or E. coli BL21 (DE3)/ΔgalU expressing WciS–His₆ (lanes 8, 9, 12 and 13) and incubated in the presence of radiolabelled (lanes 1–6, 8 and 9) or not (lanes 10–13) UDP-Gal. Native gradient PAGE (4–20% acrylamide/bisacrylamide in Tris/borate/EDTA) was performed for each sample. The migration of the ¹⁴C-galactosylated acceptor and WciS–His₆ expressed in the two strains was monitored in the gel by phosphoimaging and His₆-tag Western blotting respectively. A control was realized in the same assay conditions with only UDP-[U-¹⁴C]Gal added to the reaction mixture (lane 7). Identity of radiolabelled product (lanes 1 and 2) was confirmed by α-galactosidase gel treatment (lanes 3 and 4). SN, supernatant; MW, molecular-mass markers (sizes given in kDa).