Broad molecular weight protein standards

Ricin 2 µg, 1 µg, and 0.5 µg protein (loaded per individual well) were separated using one-dimensional SDS-PAGE in precast Novex NuPAGE 4–12% bis-tris gels (Life Technologies, Carlsbad, CA). Broad molecular weight protein standards (10–250 kDa) were used as a size reference (Bio-Rad Laboratories, Hercules, CA). Denatured proteins were visually examined using a silver stain for mass spectrometry kit (Pierce, Rockford, IL). Furthermore to assess immunoreactivity, an unstained duplicate gel consisting of separated ricin was electroblotted using the iBlot dry blotting system (Life Technologies). The protein-immobilized PVDF blot was subsequently immunodetected with an affinity purified primary goat-anti ricinus communis antibody using the anti-goat Westernbreeze chromogenic kit (Life Technologies). Of note, to ensure maximal immunoreactivity, the primary antibody (5 mg/mL; diluted in 1% nonfat dry milk) was incubated over night at room temperature on a rocking platform and the next day, the remaining steps in the Westernbreeze chromogenic kit protocol were completed.