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. 2014 May 22;6(5):1644–1666. doi: 10.3390/toxins6051644

Figure 1.

Figure 1

Chemical conjugation, purification and characterization of saporin-HRP. (A) Cross-linkage of saporin and HRP. The reaction mixture was directly analysed by SDS-PAGE. Saporin and HRP served as unconjugated controls; (B) Purification of saporin-HRP by Ni-NTA chromatography with increasing concentrations of imidazole in the elution buffer. All fractions were assessed by SDS-PAGE. Saporin-HRP was eluted in Fractions 1–3 at 62 mM imidazole (see arrow); (C) Validation of the chemical conjugation of saporin and HRP. Saporin-HRP was analysed by SDS-PAGE, and the conjugate was visualized in the gel (see arrow). Saporin-HRP was analysed by Western blot with a primary polyclonal antibody against saporin. Saporin and the conjugate (see arrow) were specifically detected in the membrane; (D) Peroxidase activity of saporin-HRP. A directly proportional correlation between absorbance and concentration was observed from 0.1 to 10 nM saporin-HRP. Each data point is the mean ± SD, n = 3; Comparison of the cytotoxicity of (E) saporin and (F) saporin-HRP in the presence of SA1641. ECV-304 cells (4000 cells/well) were treated with saporin or saporin-HRP in a concentration range from 0.01 to 100 nM alone or in combination with SA1641 (final concentration of 5 µg/mL) for 48 h. Cell viability was determined by the MTT assay. Data represents the mean ± SD, n = 4.