Figure 2.

SDS-PAGE and Western Blot analysis of both purified immunotoxins. (A) Coomassie blue stained SDS-PAGE analysis of the different pools obtained during the Ni²⁺-NTA affinity chromatography employed for the immunotoxins purification. Lines shown correspond to: FT, not retained fraction; W, washed fraction eluted with chromatography buffer (see Methods), 20 mM, washed fraction eluted with the same chromatography buffer but containing 20 mM imidazole; and the first four 1 ml fractions eluted with 250 mM imidazole. (B) Western blot analysis using rabbit anti-α-sarcin serum (left) or commercial anti-histidine tag antibody (right). α-Sarcin designates 0.1 µg of the fungal natural protein used as a control. MW corresponds to prestained Bio-Rad Precision Plus protein molecular weight standards. Images correspond to full-length gels and blots acquired and analyzed using the Gel Doc XR Imaging System and Quantity One 1-D analysis software (BioRad) or ChemiDoc-It (UVP) and VisionWorks LS, respectively. Different exposures of the blots are presented in Supplementary Fig. S1.