Anti his antibody

In the purification of E2 protein, the leaves were crushed and incubated for 30 min in an extraction buffer 1 (EB 1) consisting of 50 mM sodium phosphate (pH 8.0), 300 mM sodium chloride (NaCl), 10 mM imidazole, 0.5% (v/v) Triton X-100, 50 mM glycine, 100 mM sodium sulfite, 50 mM ascorbic acid, and 1.5% polyvinylpolypyrrolidone. With the purification of E^rns protein, an extraction buffer 2 (EB 2) consisting of 50 mM Tris-Cl (pH 8.5), 300 mM NaCl, 50 mM imidazole, 0.3% (v/v) Triton X-100, 100 mM glycine, 100 mM sodium sulfite, 1 mM phenyl methyl sulfonyl fluoride, and 1.5% polyvinylpolypyrrolidone was used. After centrifugation, the supernatant from EB 1 or EB 2 was mixed with His60 Ni Superflow Resin (Takara, Kusatsu, Japan) for 1 h at 4°C, and the bound proteins were eluted with 50 mM sodium phosphate (pH 8.0), 300 mM NaCl, and 250 mM imidazole for E2 protein or 300 mM imidazole for E^rns protein purification. The eluates were adjusted with 50 mM Tris-Cl (pH 7.2) and 150 mM NaCl. The purified proteins were treated with β-mercaptoethanol, assessed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and analyzed by Western blotting with an anti-His antibody (BioLegend, San Diego, CA, United States), anti-LOM vaccinated serum, and anti-E2 subunit vaccinated serum.