Quantifying Purified HIV-1 Viral Particles

Purified VLPs were quantified either by p24 ELISA (Innotest HIV antigen mAb, Fujirebio) following manufacturer’s instructions or by western blot. For western blot quantification, recombinant Gag protein [38 (link)] was used as standard. The standard curve started at 125 ng with 1:2 dilutions until 7.8 ng. Samples were treated as described above. Samples were denatured at 95 °C for 5 min, and proteins were separated by SDS-PAGE. After blocking, membranes were incubated with primary antibody anti-HIV-1 p24 antibody (Abcam, 1:2000) and secondary antibody Peroxidase AffiniPure Donkey anti-Mouse IgG (H+L, Jackson ImmunoResearch, 1:10,000).
The total protein content in the sample was assessed by Bicinchoninic Acid (BCA) Protein Assay (ThermoFisher Scientific).