Anti his tag monoclonal antibody

The purified recombinant ORF18R and ORF075R proteins were used to produce the rabbit anti-ORF18R and anti-ORF075R polyclonal antibodies. The monoclonal antibody (MAb) to actin was from Chemicon. Horseradish peroxidase-conjugated donkey anti-mouse and donkey anti-rabbit polyclonal antibodies were from Pharmacia. The anti -His tag monoclonal antibody was from Abcam. Goat anti-rabbit gold particles (25 nm) and goat anti-mouse gold particles (15 nm) were from Electron Microscopy Sciences. Anti-rabbit Alexa Fluor 488-conjugated antibody was from Molecular Probes (Life Technologies).

Sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and Western blotting were conducted simultaneously to detect and confirm the presence and purity of the three purified fusion proteins. Primarily, 2 μg from all the purified structural proteins were electrophoresed at 12% SDS-PAGE and protein bands were visualized by Coomassie Brilliant Blue R250 (Sigma-Aldrich, St. Louis, MI, USA). Following this step, the purified structural proteins, which were electrophoresed at 12% SDS-PAGE, were transferred to polyvinylidene fluoride (PVDF) membranes and blocked with 5% (w/v) non-fat milk for 1 h at room temperature (RT) for western blotting. After washing five times with phosphate-buffered saline-Tween solution (PBST), the membranes were incubated with anti-His-tag monoclonal antibody (diluted at 1:2000) (Abcam, Cambridge, UK) overnight at 4 °C. The membranes incubated with primary antibody was washed five times with PBST, and then incubated in rabbit anti-mouse IgG antibody (Abcam, Cambridge, UK) at dilutions of 1:5000 for 1 h at RT. The washed membranes were exposed to the ECL machine using the chemiluminescence kit (Millipore Corp., Billerica, MA, USA) to verify the recombinant proteins bands.