Anti hiv 1 p24 antibody

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).

The conjugation experiment follows a simple and rapid procedure provided along with the Streptavidin Conjugation Kit (Lightning-Link) which targets primary amine groups (e.g. lysines). Briefly, streptavidin modifiers and detection antibodies (100 μg, 1.31 mg/mL, undiluted anti-HIV-1 p24 antibody [38/8.7.47], Abcam, Cat#: ab9044) were mixed and incubated for 3 h at room temperature without light. The result solution was then quenched for 30 min and stored for future use without purification.

Co-immunoprecipitation and western blotting assays were performed as previously described [20 (link), 32 (link)]. In brief, human 293T cells were lysed with the lysis buffer (150 mM NaCl, 50 mM Tris–HCl [pH 7.5], 1 mM EDTA, 1% Triton X-100, 0.5% NP-40, plus PMSF and protease inhibitor cocktail [Sigma]) for 30 min at 4 °C. The cell lysates were clarified by centrifugation at 18,000g for 30 min at 4 °C, then mixed with anti-HA agarose beads (Sigma) and incubated at 4 °C for 4 h, followed by washing four times with cold lysis buffer and eluting in gel loading buffer. As indicated, the beads were treated with RNase mixture (DNase-free, Roche) (20 μg/ml) and incubated at 37 °C for 30 min. The immunoprecipitated samples were analyzed by SDS-PAGE and detected by western blotting. Anti-HA antibody (mouse monoclonal, Covance), anti-FLAG antibody (rabbit polyclonal, MBL), anti-GAPDH antibody (rabbit polyclonal, MBL), anti-MOV10 antibody (rabbit polyclonal, Abcam), anti-ElonginC antibody (rabbit polyclonal, Abcam), anti-Cullin 5 antibody (rabbit polyclonal, Abcam), anti-A3G antibody (rabbit polyclonal, Abcam), anti-Vif antibody (mouse monoclonal, Abcam), and anti-HIV-1 p24 antibody (rabbitpolyclonal antibodies made by our lab) were used as primary antibodies [64 (link)]. Quantity One program (Bio-rad) was used to quantify the western blotting results.