Skip to main content
. 2004 Jan 30;101(6):1519–1524. doi: 10.1073/pnas.0306533101

Fig. 5.

Fig. 5.

Pull down of untagged EmrE by EmrE-His. (A) EmrE and EmrE-His were coexpressed in vitro at different DNA ratios, as indicated (Top). The amount of plasmid coding for EmrE-His was kept constant, whereas the DNA plasmid coding for the untagged EmrE was at varying amounts and always in excess compared with the DNA plasmid coding for the tagged protein form (up to 100-fold). After the reaction was completed, 0.5-μl samples were analyzed by SDS/PAGE (Middle). Samples (20 μl) were then bound to Ni-NTA beads. Bound protein was washed three times in 0.08% DDM/Na buffer containing 25 mM imidazole and then eluted from beads and separated by SDS/PAGE (Bottom). (B) Data from seven different experiments, as described in A, were quantitated as follows. Amounts of total synthesized and pulled-down protein were estimated by using image gauge 3.46 software (Fujifilm). The ratio of pulled-down EmrE/EmrE-His was then plotted as a function of the ratio of total synthesized EmrE/EmrE-His.