Fluorescent and western blot analysis of the hypothetical ATP dependent DNA ligase domain containing protein. A. SDS-PAGE was carried out in a discontinuous slab gel under semi-denaturing conditions by omitting mercaptoethanol from the sample buffer and without boiling. About 15 μg of total protein were loaded in each lane. Gels were visualized with Bio-Rad Gel Doc EZ using the blue sample tray for GFP. Lane 1: affinity purified 6 × His-TtsfGFP from Eschericha coli; lane 2: untransformed T. thermophila total cell protein (negative control); T. thermophila with pVTtsfGFP-H induced with 0.25 μg/mL of CdCl2 for 3 h; lane 3: zero time, lane 4: 1 h, lane 5: 2 h, lane 6: 3 h. B. The 6 × His-TtsfGFP-H fusion protein purified from T. thermophila pVTtsfGFP-H clone was induced for 18 h and analyzed with western blotting by using monoclonal mouse anti-GFP antibody (1:1000). The 6 × His-TtsfGFP-H was approximately 95 kDa (Lane 1, black arrow), as expected. Many fragmented proteins were also visible. Moreover, some of the target and fragmented fusion proteins were lost during washing (Lane 2) and flow-through (Lane 3) steps of Ni-NTA affinity purification. The predicted size of the fragments based on the rare codons plus 6 × His-TtsfGFP would be approximately 36.7 kDa (Arrow 1), 48 kDa (Arrow 2) and 50 kDa (Arrow 3). The roughly 70 kDa band could be dimer of these broken fusion proteins caused by the dimerization of sfGFP (Arrow 4). M: Bio-Rad Kaleidoscope western marker.