Irdye 680cw donkey anti rabbit igg

In order to confirm the expression of luciferase-tagged VP1 proteins, transfected HEK293Ts were washed with PBS and lysed 24 hours post-transfection with RIPA buffer. Protein concentrations of the lysates were determined by BCA protein assays, and appropriate amounts of lysate were added to sample loading buffer containing 10% β-mercapto-ethanol to equal a final concentration of 1μg/mL. Samples were heated at 95°C for five minutes and separated on a 12.5% SDS-PAGE gel. The BioRad Trans-Blot Turbo system was used to transfer proteins onto a nitrocellulose membrane then blocked with 5% nonfat dry milk or BSA diluted in 1X PBS-0.05% Tween20 (PBS-T) for one hour at room temperature. Mouse anti-renilla luciferase polyclonal antibody (Millipore) and mouse anti-GAPDH polyclonal antibody (Protein Tech) were each diluted 1:1,000 in 5% nonfat dry milk or BSA in PBS-T. Anti-HuNoV strain-specific guinea pig serum was diluted 1:500. All probed membranes were incubated overnight at 4°C. Li-Cor secondary antibodies (IRDye 680CW donkey anti-rabbit IgG, IRDye 800CW goat anti-mouse IgG, and IRDye 800CW donkey anti-guinea pig IgG) were diluted 1:10,000 in 5% nonfat dry milk or BSA in PBS-T and incubated with their respective membranes for one hour at room temperature before being washed and developed using the Li-Cor Odyssey CLx imaging system.