Horseradish peroxidase conjugated anti mouse secondary antibody

In vitro translation was performed using a PURESYSTEM classic II (BioComber, Tokyo, Japan). For the RNA template for in vitro translation, either a +1-peuA′-flag RNA (30 pmol)/fur-flag RNA (3 pmol) mixture or a +39-peuA′-flag RNA (30 pmol)/fur-flag RNA (3 pmol) mixture was used. The PURESYSTEM reaction mixture (20 µl) was incubated at 37°C for 2 h, and the reaction was then terminated by adding an equal volume of 2× SDS-PAGE sample buffer. The samples were separated on a 15% SDS-polyacrylamide gel, and the protein bands were transferred to a Clear Blot Membrane-P (Atto, Tokyo, Japan). The membrane was blocked with Tris-buffered saline with Tween 20 (TBST) containing 0.3% skim milk and incubated overnight at 4°C with mouse anti-FLAG M2 antibody (Sigma) diluted 1,000-fold with blocking solution. The membrane was then washed four times with TBST, incubated for 1 h at room temperature with horseradish peroxidase-conjugated anti-mouse secondary antibody (GE Healthcare), diluted 20,000-fold with blocking solution, and washed four times with TBST. Immunoreactive bands were detected with an ECL Select Western Blotting Detection Reagent (GE Healthcare) and visualized with a LAS-3000 gel imager (Fujifilm, Tokyo, Japan)

Proteins of the cell lysates and cell culture supernatants were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The blots were blocked with 5% milk in Tris-buffered saline with 0.1% Tween-20 for 1 h, then probed with rabbit polyclonal anti-β-actin antibody (diluted 1:4000; Abcam, Cambridge, UK), mouse monoclonal anti-HBs antibody (diluted 1:2000; Cosmo Bio, Tokyo, Japan) or mouse monoclonal anti-HBV pre-S1 antibody (diluted 1:4000; Beacle, Kyoto, Japan) at 4 °C overnight. The immunoblots were then probed with horseradish peroxidase-conjugated anti-mouse secondary antibody (diluted 1:4000; GE Healthcare, Amersham, UK) and visualized using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare). Protein amounts were determined by densitometry.

Total of 5*10¹⁰ particles of all samples were prepared with 6x Laemmli sample buffer with 10% β-mercaptoethanol and run on 4–20% gradient SDS-PAGE (Mini-Protean TGX Precast protein gel, Bio-Rad, Hercules, USA). For Western blot analysis, proteins were transferred to PVDF membrane (Immobilon-P, Merck Millipore, Burlington, USA) at 200 mA for 90 minutes using wet transfer. Nonspecific binding was blocked by with 3% BSA in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 hour followed by primary antibody incubation anti-TSG101 (Clone 51/TSG101 BD Biosciences, 1:500), anti-CD41 (clone SZ22, Beckman Coulter, 1:2000), or anti-CD9 (clone C-4, Santa Cruz Biotechnology, Dallas, USA, 1:500) overnight at 4˚C. The membranes were washed three times with TBST followed by incubation with horseradish peroxidase–conjugated anti-mouse secondary antibody in dilution 1:3000 (GE Healthcare Bio-Sciences AB). After washing, the signal was detected using Bio-Rad Clarity Western ECL Substrate (Bio-Rad) and imaged using LAS3000 imaging system (Fuji, Minato, Japan).