Figure 5.
The rapid dilution method produces properly folded, monomeric mVDAC1, as assessed by size exclusion chromatography (SEC) and circular dichroism
(A) SEC profile of refolded mVDAC1 collected on a Cytiva Superdex 200 Increase 10/300 GL column. Peaks 1, 2, and 3 elute at 8.6 mL, 12.4 mL, and 14.9 mL, respectively.
(B) Coomassie-stained SDS-PAGE of refolded mVDAC1 eluted from the size exclusion column. Peaks 1–3 from (A) are indicated above the gel. Equivalent volumes of each peak are loaded. The faint double band observed in P1 is due to the crosslinking of the two cysteines in the improperly folded forms, leading to faster migration on the gel.8
(C) Circular Dichroism spectrum typical of refolded mVDAC1 showing high beta-sheet content. Data were collected on a Jasco-815 spectrometer in PBS with 0.1% LDAO.