Fig. 7. NPGal protection against [¹⁴C]NEM labeling of (A) pseudo-wild-type single-Cys148 LacY and (B) mutant E269D/single-Cys148. RSO membrane vesicles containing single-Cys148 LacY with a biotin acceptor domain at the C-terminus were incubated in 100 mM KP_i pH 7.5 with 0.5 mM [¹⁴C]NEM (40 mCi/mmol) for 5 min in the absence or presence of the indicated concentrations of p-NPGal. Reactions were quenched with DTT, and biotinylated LacY was solubilized and purified by affinity chromatography on monomeric avidin. Samples were separated on a sodium dodecylsulfate–12% polyacrylamide gel and labeled protein was visualized by autoradiography. Labeling was quantitated with a Storm 860 PhosphorImager, and labeling in the presence of given concentrations of NPG is expressed as percent labeling observed in the absence of sugar.