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. 2011 Oct;193(19):5242–5251. doi: 10.1128/JB.05429-11

Fig. 3.

Fig. 3.

His-YidD is localized at the IM. (A) Subcellular fractionation of MC4100-A containing pEH3His-YidC. Cells were grown in LB medium and induced with 100 μM IPTG for 1 h and fractionated, and the fractions were analyzed by Western blotting using antisera against the His tag and the control IMP Lep. T, total cell sample; D, cellular debris (pellet from low-speed centrifugation); S, soluble fraction (supernatant of high-speed centrifugation); M, membrane fraction (pellet of high-speed centrifugation). Four times the amount of material of the total cell sample was loaded for fractions D, S, and M. (B) Membranes from MC4100-A expressing His-YidD were separated by sucrose gradient centrifugation. Fractions were analyzed by Western blotting using antisera against the His tag, the control IMP Lep, and the control OMP TolC. (C) Membranes from MC4100-A expressing His-YidD were treated with 0.5% Sarkosyl to solubilize the IMs and centrifuged to collect the OMs. Samples of the fractions, derived from equal amounts of cell material, were analyzed by SDS-PAGE and Western blotting using antisera against the His tag, the control IMP Lep, and the control OMP TolC. M, total membranes; IM, inner membrane (supernatant of Sarkosyl extraction); OM, outer membrane (pellet of Sarkosyl extraction).