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. 2014 Jun 18;9(6):e99884. doi: 10.1371/journal.pone.0099884

Figure 4. Confirmation of cloned construct, expression, purification and Western blotting of recombinant wBm-MurA.

Figure 4

A: The cloned gene within the expression vector pET28a was checked by restriction-digestion. Lane 1, molecular size marker (GeneRuler 1 kb Plus DNA Ladder, Thermo Scientific); lane 1–2, Restricted plasmid (insert-1278 bp); lane 3, un-restricted construct (pET28a containing the insert). B: Coomassie-stained SDS-polyacrylamide gel of recombinant wBm-MurA over-expressed in Rosetta(DE3)pLysS E. coli strain with a His tag fusion protein. Lane 1, molecular mass markers (Puregene 4 Color Prestain protein ladder, Genetix); lane 2, uninduced E. coli lysate; lane 3; E. coli lysate after 22 h induction with 0.2 mM IPTG at 24°C; lane 4, flowthrough after passing the supernatant through an Ni-NTA column; lane 5, 10 column volumes eluted with wash buffer containing 60 mM imidazole; lane 6, purified wBm-MurA recombinant fusion protein eluted with wash buffer containing 300 mM imidazole. C: Western blot developed with diaminobenzidine using mouse anti-His monoclonal antibody as primary antibody and HRP-conjugated anti-mouse IgG (lane 2) as secondary antibody; lane 1, molecular mass markers (Puregene 4 Color Prestain protein ladder, Genetix).