FIGURE 3.
Western blot analysis. C41(DE3)pLysS transformed with the empty pET-14b vector (lanes 2 and 3) or the TM4-Cx45CT plasmid (lanes 5 and 6). Lane 8 contains the TM4-Cx45CT after purification. Lane 1 contains the Precision Plus Protein All Blue Standards molecular mass marker (Bio-Rad) and lanes 4 and 7 are blank. Samples were collected just prior to (lanes 2 and 5) and 4 h after IPTG induction (lanes 3 and 6). A total of 500 μL samples with an Abs600nm at 0.5 were pelleted and resuspended in 30 μL of 6× SDS loading buffer. Equal amounts of total protein (7 μL) were ran on a 15% SDS-PAGE gel. (A) Coomassie Blue stained gel is shown as a reference. Western blot analyses were performed using either (B) anti-His6, or (C) anti-Cx45 primary antibodies. The expected molecular mass of the TM4-Cx45CT is ~22 kDa, which is indicated by the arrow. Of note, in (B) lane 8, the anti-His6 primary antibody also reacted with a ~20 Da protein. Although not present in the (A) 15% SDS-PAGE gel or reactive with the (C) anti-Cx45 primary antibody, we speculate the doublet is caused by proteolysis of the TM4-Cx45CT.