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. 2013 Nov 6;8(11):e78272. doi: 10.1371/journal.pone.0078272

Figure 7. Blue native (BN) PAGE, SDS PAGE and WB analysis of the P66 complex.

Figure 7

The complete outer membrane fraction of B. burgdorferi B31 was applied to a 4–16% BN PAGE. A 460 kDa band that reacted to the P66 antibody was extracted from the gel and loaded again in a 4–16% BN PAGE (BN/BN PAGE). This gel was stained with silver nitrate (SN) and subjected to a western blot (WB) against P66 (A). The 460 kDa band was extracted from the gel using detergents and resolved again under denaturing conditions in glycine and tricine SDS PAGE (B). A whole lane from the BN/BN PAGE containing the P66 complex was resolved in a second dimension SDS PAGE (C). The elution of the 460 kDa band from the BN/BN PAGE was tested in planar bilayers for pore-forming activity. Step-like increases in the conductance of the membrane were observed after adding the sample to the salt solution (D, left panel) and at least 100 insertional events were summarized in a histogram (D, right panel).