Fig 2. Western blot and BN-PAGE analyses of peak I and II. (A) SDS-PAGE of peak I (lane 1) and peak II (lane 2) under DTT. (B) Western blot of peak I and peak II, with DTT, using Sup35 sequence-specific antibody. (C) BN-PAGE analysis of peak I. Peak I + DTT and Peak II + DTT all had the same amount of total protein in Figures 2A and 2B. The amount of protein in Figure 2C was twice as much as that in Figure 2A. Lanes M in Figures 2A and 2C: molecular weight protein marker comprising 116 kDa, 66.2 kDa, 45 kDa, 35 kDa, 25 kDa, 18.4 kDa, and 14.4 kDa.
Due to the relatively high molecular weight of Sup35 (685 amino acids, 5 cysteine residues, 76.6 kDa as calculated from the amino acid sequence), Sup35 fused with histidine (His-tag) in the C-terminal was chosen for the elimination of partially translated Sup35 (Fig S1). Western blot with sequence-specific antibody shows that the band between 116 kDa and 66.2 kDa was the Sup35 monomer band, while the other dissociated bands had no signal (Figs 2A and 2B). This indicates that peak I comprises a monomer and other proteins, with the whole complex interacting with Ni2+ affinity chromatography through the His-tag. Meanwhile, peak I exhibited a monomer band and high molecular weight bands (including a band at the loading slot) in blue native polyacrylamide gel electrophoresis (BN-PAGE) and could not be disaggregated to form clear X1 and Omp F bands (Fig 2C). This was the foundation of the Sup35 monomer SEC purification (Fig 3). All these conclusions were prerequisite for the Sup35 monomer and complex research below.