HIV-1 Env Protein Western Blot

Western blot was performed as described previously [36 (link)]. Briefly, VLPs and recombinant proteins were solubilized in RIPA Buffer (Sigma, St. Louis, MO) and then in 2X Laemmli Buffer (Bio-Rad, Hercules, CA). After boiling the samples for5 minutes, we loaded them into a 10% SDS-PAGE gel and proceeded with electrophoresis for 2 hours at 100 volts. The protein was transferred to nitrocellulose for 2 hours at 90 volts, 4°C. Ponceau S stain (Sigma, St. Louis, MO) was used to verify protein transfer and the membrane was incubated overnight at 4°C with primary antibody, human monoclonal antibody to V3 of HIV-1 Env (447-52D; NIH AIDS Reagent Program). The following day, the membrane was washed 3 times in TBST (Tris-buffered saline plus Tween 20) and incubated for 2 hours at room temperature with anti-human HRP-conjugated secondary antibody (Southern Biotech, Birmingham AL). The secondary antibody was removed and the membrane washed 5 times with TBST, incubated with chemiluminescent substrate (GE, Schenectady, NY), and exposed to X-ray film (Denville Scientific, Metuchen, NJ). The film was developed with a Kodak X-GMAT 2000 (Eastman Kodak, Rochester, NY).