Immuno blot pvdf membrane

An Immuno-Blot PVDF membrane (Biorad) was activated and calibrated with pure methanol and PBS for the dot blot. Drops of 2 µL were placed on the membranes (3 drops per piece) containing HBsAg (1 µg/mL) either diluted in PBS (non-reducing condition) or heated for 10’ at 95°C in dot blot buffer (0.125 M Tris-HCl, 4% SDS, 20% Glycerol, 10% β-mercaptoethanol) (reducing condition). After complete drying, the membranes were blocked with 5% milk in TBS-T buffer for 1h at RT. Membranes were then transferred into 5% milk TBS-T solutions containing the primary antibodies 4D06, 4D08, or HB-1 with a concentration of 1 µg/ml. After incubation for 1.5h at RT, the membranes were washed 3x for 10’ in TBS-T buffer and placed into 5% milk in TBS-T buffer containing secondary antibody goat anti-human IgG HRP (Sigma Aldrich) diluted 1:10,000 for 1h at RT. After final washing, an ECL detection solution (GE Healthcare) was added to develop and analyze the blots.
The HBsAg Western blot was performed similarly. HBsAg was heated for 20’ at 95°C in dot blot buffer and loaded onto a 12% SDS-PAGE gel. Separated proteins were wet-blotted onto an Immuno-Blot PVDF membrane (Biorad) and blocked in the same way as described above for the dot blot. The antibodies were used at 1 µg/mL diluted in 5% milk in TBS-T buffer. All further steps were performed as in the dot blot.

Total protein from the C2C12 cells was extracted with a lysis buffer containing 50 mM HEPES (pH: 7.6), 150 mM NaCl, 10 mM EDTA, 10 mM Na₄P₂O₇, 10 mM NaF, 2 mM Na₃VO₄, 1% (vol/vol) NP-40, 1% (vol/vol) Na-deoxycholate, 0.2% (wt/vol) SDS, and 1% (vol/vol) complete protease inhibitor cocktail (Nacalai Tesque Inc. Kyoto, Japan). Protein concentrations were measured using a Protein Assay Bicinchoninate Kit (Nacalai Tesque Inc. Kyoto, Japan). Before SDS-PAGE, an aliquot of the extracted protein solution was mixed with an equal volume of the sample loading buffer containing 1% (vol/vol) 2-mercaptoethanol, 4% (wt/vol) SDS, 125 mM of Tris-HCl (pH: 6.8), 10% (wt/vol) sucrose, and 0.01% (wt/vol) bromophenol blue. The mixture was then heated to 37℃ for 30 min. Then, 10 mg of protein was separated on SDS-polyacrylamide gels and electrically transferred to an ImmunoBlot PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA). The blot was blocked with the Blocking One (Nakalai Tesque Inc. Kyoto, Japan) for 1 h at room temperature and incubated with primary antibodies overnight at 4℃ in TBS containing 0.1% Tween-20. The signals were detected using the Immunostar Zeta or LD (Wako Chemicals. Osaka, Japan), quantified using the C-Digit (LI-COR Biosciences, Lincoln, NE, USA), and expressed as arbitrary units.