Irdye 800cw goat anti human

Expression, refolding, and identity of recombinant proteins were assessed by SDS-PAGE and Western blot analysis using Invitrogen NuPAGE 4–12% Bis-Tris mini-gels (Carlsbad, CA) under reducing conditions. For SDS-PAGE, gels were stained with SimplyBlue SafeStain (Invitrogen). For Western blot analysis, proteins were transferred onto nitrocellulose paper (Invitrogen), blocked with Odyssey blocking buffer (LI-COR Biosciences) overnight at 4°C. The following day, the blot was probed simultaneously with serum from a rabbit immunized with E2wt (1∶8000) and the human mAbs 2F5 or 4E10 (1∶20,000) for 1 h at room temperature. Primary Abs were prepared in Odyssey blocking buffer 1∶1 with 1xPBS, 0.2% Tween-20. Blots were washed 5 times with 0.1% Triton X-100, 1xPBS. Secondary Abs IRDye 680 Goat anti-Rabbit and IRDye 800CW Goat anti-Human (LI-COR Biosciences) were used at 1∶15,000, diluted in Odyssey blocking buffer 1∶1 with 1× PBS, 0.2% Tween-20, 0.02% SDS. Membranes were scanned using the LI-COR Odyssey Infrared Imaging System (LI-COR Biosciences) to allow simultaneous two-color detection of E2 and the HIV-1 Envelope 2F5 or 4E10 epitopes. Integrated intensities were used in conjunction with protein concentrations determined by NanoDrop (NanoDrop Technologies, Wilmington DE) to calculate protein purity and concentration.

Ten thousand cells per well of C6/36 cells were seeded in a 96-well plate overnight. Virus samples were diluted in 10-fold serial dilutions in RPMI supplemented with 2% FBS and 50 μL from each dilution was added into each well containing the cells, and incubated at 28 °C. Two-hour post infection, 150 μL of overlay media (2× M199 medium, 2% FBS, 50 U/mL penicillin, 50 µg/mL streptomycin, 2% carboxymethyl cellulose) was added. Seventy-two hours after infection, the overlay was removed and 100 μL of ice-cold 80% acetone in PBS was added and incubated at −20 °C for 30 min to fix the cells, and then air-dried. The plate was first blocked for 1 h at 37 °C with 100 µL of blocking solution (KPL, SeraCare), followed by the addition of primary 50 µL of mAb 4G4 for 1 h at 37 °C. Plates were then washed three times with PBS/T. Secondary antibody (IRDye 800CW Goat anti-human, LI-COR, USA) at 1:2500 dilution was added (50 µL/well) and incubated for 1 h at 37 °C in the dark. Plates were washed three times, air-dried and then kept in the dark at room temperature until ready for imaging. Plates were scanned using the LI-COR Biosciences Odyssey Infrared Imaging System. Viral titres from IPA are expressed as focus-forming units per mL. The results were plotted in GraphPad Prism (v. 8.1.2, La Jolla, CA, USA).