Irdye 800cw conjugated goat anti mouse secondary antibody

Vero cells (3×10⁶) were seeded in 60-mm dishes and inoculated with 2.5 pfu per cell for 14 hours. The cells were collected by treatment with 5mM EDTA (prepared in 1× PBS) and whole-cell lysates prepared by boiling for 5 minutes in Laemmli sample buffer [92 (link)]. Lysates equivalent to 1×10⁵ cells per lane were resolved by SDS/PAGE (10% gels), and transferred onto nitrocellulose membranes (Millipore). After incubation in blocking buffer [5% Difco skimmed milk (BD Biosciences) in TBST (Tris-buffered saline with 0.2% Tween-20)], the membranes were incubated overnight with mouse monoclonal antibodies against ICP0, glycoproteins gB, gC, gD, gE, and gG, and major capsid protein (VP5) diluted to 1:1,000 in blocking buffer. The membranes were washed in TBST and incubated for 1 hour with IRDye-800CW-conjugated goat anti-mouse secondary antibody (Li-Cor Biosciences) diluted 1:20,000 in blocking buffer. After additional washes with TBST, the proteins of interest were visualized on an Odyssey Infrared Imaging System (Li-Cor Biosciences). Equal loading of the samples was verified by re-probing the membranes with anti-β-actin mouse mAb diluted 1:2,000 in blocking buffer.