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. 2013 Oct 4;8(10):e75028. doi: 10.1371/journal.pone.0075028

Figure 2. Purification, stoichiometric characterization and secondary structure analysis of YsaL/Ye3555.

Figure 2

(A) SDS PAGE analysis of refolded YsaL-His after Ni-NTA affinity chromatography: M-Marker, UI- Uninduced, I- induced, E1 and E2- Ni-NTA eluates. (B) Chemical crosslinking profile of refolded YsaL-His in SDS-PAGE using 1.5% Gluteraldehyde - M- Marker Ctrl- Control, C- crosslinking of YsaL-His using 1.5% Gluteraldehyde incubated for 5 min. (C) Size exclusion chromatography (SEC) profile of refolded YsaL-His in pre-calibrated Superdex 200 hi-load16/60 column (GE Healthcare). Molecular weight standards are indicated with triangles: Thyroglobulin (T) –669 kDa, Ferritin (F) –440 kDa, Aldolase (A) –158.4 kDa, Ovalbumin (O) –48 kDa, Carbonic anhydrase (C) –29 kDa and Ribonuclease A (R) –14 kDa. SDS PAGE analysis of Gel filtration profile: M – marker, P1 (peak1) - YsaL-His (inset). (D) Molecular mass estimation from size exclusion profile of refolded YsaL-His using the above known standards. Refolded YsaL-His was dimeric (∼48 kDa). (E) Far UV-CD (Circular dichroism) spectra of refolded YsaL-His (HT) and refolded untagged (UT) YsaL.