Hrp conjugated protein g

Culture supernatants of the vector-producing cells were filtered through a 0.45-μm membrane (Millipore), and centrifuged at 16,114 × g through 20% sucrose for 5 h to collect virion pellets. Cell lysates and virion pellets were subjected to SDS polyacrylamide gel electrophoresis with or without Phos-tag reagent (Kinoshita et al., 2006 (link)), and proteins were transferred onto a PVDF membrane. Membranes treated with rabbit anti-HIV-1 p24 (BioAcademia or ZeptoMetrix), sheep anti-HIV-1 gp120 (provided by Dr. T. Murakami), or rabbit anti-ezrin antibody (Santa Cruz Biotechnology) then were treated with HRP-conjugated protein G (BioRad) to detect the proteins. Membranes treated with mouse anti-VSV-G epitope (Sigma-Aldrich) and mouse anti-actin antibodies (Santa Cruz Biotechnology) were treated with HRP-conjugated anti-mouse IgG (BioRad) as the secondary antibody. Antigen proteins were visualized using the Clarity Western ECL substrate (BioRad).

COS7, Vero, or 293T cells were transfected as indicated or treated with concanamycin A (CMA) or MG-132 (Sigma-Aldrich). Cells were transfected with the HIV-1 vector construction plasmids. The transfected cells were cultured for 24 h, and washed with medium to remove the transfection reagent and plasmids. The cells were additionally cultured for 24 h, and cell lysates and culture supernatants were prepared from the cells. Cell lysates were subjected to SDS polyacrylamide gel electrophoresis (BioRad). Proteins were transferred to a PVDF membrane (Millipore). The membrane was treated with indicated first antibodies. As the first antibodies, rabbit anti-HIV-1 p24 (BioAcademia), goat anti-HIV-1 gp120 (Fitzgerald), goat anti-MLV p30 (ViroMed Biosafety Laboratories), mouse anti-actin (Santa Cruz), mouse anti-VSV-G (Sigma-Aldrich), and goat anti-ezrin (Santa Cruz) antibodies were used. When mouse antibodies were used as the first antibody, HRP-conjugated anti-mouse IgG (BioRad) was used. When the membrane was treated with a rabbit or goat antibody, HRP-conjugated protein G (BioRad) was used. Intensities of protein bands were measured by the ImageJ software.

The following IgG detection probes were used for immunoassays: HRP-conjugated Protein A (#95217)/ HRP-conjugated Protein G (#64256837) were purchased from Bio-Rad® (Hercules, CA, USA). HRP-conjugated Protein A (#101023)/HRP-conjugated Protein G (#101223) were purchased from Thermo-Fisher (Waltham, MA USA). HRP-conjugated anti-mouse IgG (#A4416, Sigma), HRP-conjugated anti-cattle IgG (#A5295, Sigma), HRP-conjugated anti-dog IgG (#A6792, Sigma), HRP-conjugated anti-rat IgG (#A9037, Sigma), HRP-conjugated anti-horse (#A6917, Sigma), HRP-conjugated anti-pig IgG (#A5670, Sigma) and HRP-conjugated anti-human IgG (#A170, Sigma) were used in this study. ELISA kits for visceral leishmaniosis (REF: #VET043-1) and equine infectious anemia (REF: #VET037-1) were kindly provided by Bioclin®. Scienco Biotech kindly provided tetramethylbenzidine (ONE STEP-TMB Linear, Scienco Biotech).