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. Author manuscript; available in PMC: 2014 Sep 17.
Published in final edited form as: Biochemistry. 2013 Aug 30;52(37):6568–6574. doi: 10.1021/bi401026d

Fig. 2. SDA of single-Cys S311C.

Fig. 2

RSO membrane vesicles containing single-Cys C154V/S311C (○) or single-Cys C154G/S311C (●) were incubated with 400 μM TAM1 for the given times at room temperature in the absence of sugar. After terminating the reaction and removing the remaining TAM1, the vesicles were labeled with 40 μM TMRM at 0°C in the presence of 5 μM NPG for 30 min. LacY was solubilized and purified in DDM by monomeric avidin chromatography and subjected to SDS-PAGE. The gel was scanned for TMRM fluorescence (bands above the dotted lines) and silver stained for protein (bands below the dotted lines). Relative TMRM labeling was calculated semi quantitatively as described in Materials and Methods. Relative TMRM labeling of the 0-sec TAM1 labeling point was set to 1. V, C154V background; G, C154G background.