Fig 3. Dissection of peak I and subsequent analysis.(A) Size exclusion chromatography (SEC) curve of peak I. Imidazole was
removed from the peak I sample using dialysis. 45.5 min, 67.2 min, and
79.2 min represented the different retention times of the fractionated
species. Buffer C was used here (20 mM Tris, pH 8.0, 1.2 M NaCl). (B)
SDS-PAGE for 45.5 min, 67.2 min, and 79.2 min fractions in Figure 3A.
The bands in black, blue, and red frames were selected for mass
spectrometry. (C) SDD-AGE of peak I SEC fractions. (D) Transmission
electron microscopy (TEM) of 45.5 min species from Figure 3A. Lane M in
Figure 3B: molecular weight protein marker comprising 100 kDa, 70 kDa,
50 kDa, 40 kDa, and 30 kDa. The buffer used here was composed of 20 mM
Tris, pH 8.0, 1.2 M NaCl, and 10 mM DTT. For HiLoad Superdex 200 pg
16/60 size exclusion chromatographic column (1 mL/min), the gel phase
distribution coefficient (Kav) of Conalbumin (75 kDa),
Aldolose (158 kDa) and Ferritin (440 kDa, Horse Spleen) in Gel
Filtration Calibration kit LMW (28-4038-42) were close to 0.38, 0.26 and
0.10, respectively.