Influenza A Virus M1 Oligomerization Figure 6. The characterization of oligomerization of C-terminal domain. (A) After affinity chromatography, purified M1C without the N- terminal GST tag (affinity), yielded a single peak (1) on a Superdex 200 column. The samples were also analyzed by Tricine-SDS-PAGE. (B) The 17.3-ml fraction of C-terminal domain from panel A was collected, concentrated and re-applied to gel filtration column. The elution positions of M1C with different loading concentrations and at different pHs are shown. (C) The panel showed the sedimentation coefficient distributions c(s) of purified M1C with the concentration of 2.8 mg/ml at pH 7.4. (D) The molecular mass distribution of M1C derived from c(s) data of panel C. (E) Cross-linking of M1C. Lane 1 is M1C in the absence of cross-linker, and lane 2 is the cross-linked M1C dimer. Lane 3 and 4 are M1N without/with treated by cross linking agent, respectively. The center lane is the molecular mass marker. (F) Pull-down assay was carried out at pH 7.4. Purified M1C from gel filtration was bound to GST-beads bound GST-M1 in the binding buffer (pH 7.4). The hisM1 bound with GST-M1 was used as positive control. M1C or hisM1 incubated with GST was used as negative control. The bound M1C were examined by anti-M1 mono-antibody. doi:10.1371/journal.pone.0037786.g006 PLoS ONE | www.plosone.org 8 May 2012 | Volume 7 | Issue 5 | e37786