Amplification and purification of TbWee1-6xHis fusion protein to produce anti-TbWee1 polyclonal antibodies.

<p>(A) SDS–PAGE analysis of lysates obtained from <i>Escherichia coli</i> IPTG.induced (I) and non-induced (NI) cell cultures transformed with pDEST17-TbWee1. Upper panel: Coomassie Brilliant Blue staining. Lower panel: Western-blot analysis with anti-histidine tag antibody (1:5000). The arrow indicates the position of the ~70 kDa recombinant protein. (B) Purification steps of rTbwee1-6xHis fusion protein were monitored by 12 % SDS-PAGE analysis. The lysate was loaded onto a Ni-agarose column. Flow-through (F), washes (W, 1–3), and eluted fractions (250 mM imidazol, E 1–3) were analyzed by 12% SDS-PAGE stained with Coomassie Brilliant Blue. (C) Western blot analysis of rTbWee1 expression with polyclonal anti-TbWee1 antibody. Protein extracts of the <i>T. brucei</i> bloodstream form (lane 1), the <i>T. brucei</i> procyclic form (lane 2) and the recombinant TbWee1-6xHis (lane 3) were separated by 10 % SDS-PAGE and electroblotted on nitrocellulose membrane. Blots were incubated with anti-TbWee1 polyclonal antibody (1:1000) and revealed by chemiluminescence.