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. 2010 Dec 10;30(2):439–449. doi: 10.1038/emboj.2010.321

Figure 6.

Figure 6

Biochemical characterization of NhaP1. (A) Western blot of everted vesicles used in the activity assay. 30 μg total protein was loaded per lane and detected with an antibody against the C-terminal helix of NhaP1 coupled to horseradish peroxidase. (B) Ni-NTA-affinity purified WT and Δ22 protein (5 μg) separated on a 12% gel and stained with Coomassie brilliant blue. (C) Western blot of Ni-NTA-affinity purified WT and Δ22 protein (5 μg) transferred to a PVDF membrane, immunostained with anti-myc antibody coupled to horseradish peroxidase. (D) Blue-native gel electrophoresis of WT (30 μg) and Δ22 (20 μg). Samples were run on 4–16% Bis/Tris native-PAGE and destained. The NhaP1 dimer is indicated with an asterisk, the monomer by a diamond. WT: NhaP1wt-Myc-His; WT2: NhaP1wt-His; Δ22: NhaP1Δ22-Myc-His; Δ27: NhaP1Δ27-Myc-His; –: untransformed KNabc cells; M: prestained protein marker (Fermentas); M1: HMW protein mix for native electrophoresis (GE Healthcare).