Westernbright sirius western blotting detection kit

Virus producer cells were lysed in 1× SDS loading buffer containing 5% 2-mercaptoethanol. Purified virions were diluted in an equal volume of 2× SDS loading buffer containing 5% 2-mercaptoethanol. p24 was detected by Western blotting with an anti-HIV-1 p24 Gag mouse monoclonal antibody (31 (link)) (catalog no. 6458; NIH AIDS Reagent Program). Expression of the β-actin loading control was detected with anti-β-actin antibody sc-4778 (Santa Cruz). After being probed with the primary antibodies, the blots were washed and incubated with anti-mouse IgG–horseradish peroxidase (A9044; Sigma-Aldrich). The membranes were washed again and incubated with the WesternBright Sirius Western blotting detection kit (K-12043-D20; Advansta). Chemiluminescence was visualized with the G:BOX Imaging System and GeneSys software (Syngene). p24 and β-actin levels were then quantified with GeneTools (Syngene)

Prior to immunoblot analysis, cell lysates were diluted in 2X loading buffer (62.5 mm Tris–HCl (pH 6.8) (Sigma-Aldrich, USA), 20% glycerol (Invitrogen, CA, USA), 2% SDS (Invitrogen, NY, USA), 4% β-mercaptoethanol (Sigma-Aldrich, CA, USA), 0.008% bromophenol blue (Bio-rad, CA, USA)) and boiled at 100°C, 5 min. Proteins were then resolved via SDS-PAGE (BioRad, USA) and transferred onto nitrocellulose (GE Healthcare, USA) membranes at either 100 V for 2 h or 30 V for 16 h. The membranes were then blocked with 5% non-fat milk in TBS-T buffer (25-mm Tris–HCl, pH 7.4, 137-mm NaCl, 2.68-mm KCl and 0.05% Tween-20) (Sigma-Aldrich, USA) for at least 1 h at room temperature (RT). The membranes were then incubated with desired primary antibodies (Table 2) and rotated in 4°C overnight. Subsequently, the membranes were washed in TBS-T buffer and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Table 2) for 1–2 h at RT. HRP signals, which correspond to protein levels, were detected by various ECL detection reagents (ECL Western Blotting Substrate (Pierce, ThermoScientific, USA), Amersham ECL Western Blotting Detection Reagents (GE Healthcare, USA) and WesternBright Sirius Western Blotting detection kit (Advansta, CA, USA)).