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. 2017 Feb 22;12(2):e0172529. doi: 10.1371/journal.pone.0172529

Fig 6. Protein analysis of the talon metal affinity chromatography purification of PelB-Vpu.

Fig 6

Protein fractions were subjected to SDS-PAGE followed by silver staining (A) and immunoblotting (B). Detergent (βDDM)-soluble fraction containing PelB-Vpu (lane 1) was loaded onto a column containing Talon resin that was pre-equilibrated with 20 mM HEPES, pH 7.5, supplemented with 500 mM NaCl, 5 mM imidazole, and 0.02% βDDM. Unbound proteins in the flow through were collected (lane 2) and the column was washed consecutively with four different buffer solutions (20 mM HEPES, pH 7.5): W1 (lane 3, buffer plus 500 mM NaCl), W2 (lane 4, buffer plus 500 mM NaCl and 0.02% βDDM), W3 (lane 5, buffer plus 250 mM NaCl and 0.02% βDDM), and W4 (lane 6, buffer plus 250 mM NaCl, 0.02% βDDM and 10 mM imidazole). Pure PelB-Vpu was eluted with W4 buffer supplemented with 300 mM imidazole (lane 7, arrows).