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. Author manuscript; available in PMC: 2008 Sep 1.
Published in final edited form as: Exp Parasitol. 2007 Mar 27;117(1):99–105. doi: 10.1016/j.exppara.2007.03.010

Fig. 4.

Fig. 4

TcRBP19 protein is a low level differentially expressed protein. Protein extracts were separated by electrophoresis in 15% SDS-polyacrylamide gels and electro-transferred onto Hybond-C membranes (GE Healthcare) following standard procedures. Blots were blocked by incubation in 5 % skim milk powder in PBS and where then incubated with the purified polyclonal anti-TcRBP19 antibody in PBS-Tween buffer for 1 hour at room temperature. Bound antibodies were detected using peroxidase conjugated AffiniPure goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch) diluted 1/7,500, with the color reaction developed using 5 mg of DAB (Sigma) in 10 mL 0.05 M Tris pH 7.6 added with 10 μL 30% H2O2. Alternatively, after extensive washings in PBS-Tween-5 % skim milk, the filter was incubated with [125I] labeled protein-A (approx 5.105 cpm/mL) (GE Healthcare), extensively washed and exposed to X-ray film (Hyperfilm, GE Healthcare, UK). (A) Life cycle of wild type parasites. Total protein extracts corresponding to 5×106 parasites per lane. Lane 1: 200ng of purified TcRBP19 recombinant protein; lane 2: epimastigotes 3 days; lane 3: epimastigotes 5 days; lane 4: 24 hours of TAU3AAG nutritional stressed epimatigotes; lane 5: metacyclic trypomastigotes; lane 6: amastigotes Molecular weights are indicated. (B) TcRBP19 over expressing epimastigotes. Total protein extracts corresponding to 107 parasites per lane. Lane 1: epimastigotes wt; lane 2: epimastigotes transfected with pTEX; lane 3: epimastigotes transfected with pTEX-TcRBP19; lane 4: epimastigotes transfected in replica with pTEX-TcRBP19. Molecular weights are indicated. (C) Western blot using [125I] labeled protein-A. Total protein extracts corresponding to 108 parasites per lane. Lane 1: epimastigotes; lane 2: metacyclic trypomastigotes; lane 3: axenic amastigotes; lane 4: trypomastigotes; lane 5: cellular amastigotes; lane 6. epimastigotes transfected with pTEX-TcRBP19. (D) Western blot using the same extracts of panel C using anti-PEPCK antibody as internal control.