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

Fig. 1.

Fig. 1.

In vitro synthesis, expression, and localization of EmrE and its homologues. (A) GFP (lanes 1 and 2) and EmrE-His (lanes 3 and 4) were synthesized by using the rapid-translation system 100 E. coli HY kit and then incubated with Ni-NTA beads. After purification and elution from the beads, samples equivalent to 16.5% (lanes 1 and 3) and 50% (lanes 2 and 4) of the reaction volume were separated by SDS/PAGE and detected by Coomassie blue staining. (B) Addition of detergent mobilizes synthesized EmrE from an insoluble fraction to a soluble fraction. We synthesized 35S-labeled EmrE-His with and without addition of 0.08% DDM, as indicated. Samples were taken from the supernatant immediately after reaction (lanes 1 and 2) and after a centrifugation step of 10 min at 20,800 × g (lanes 3 and 4) and pellet fraction (lanes 5 and 6). (C) EmrE homologues BPsmr, TBsmr, Hsmr, and Psmr were synthesized in vitro. A 20-μl sample was then loaded on Ni-NTA beads. Proteins were purified and eluted from beads, separated by SDS/PAGE, and detected after Coomassie blue staining.