Trans blot sd semi dry transfer cell device

The reactivity of the hybridoma supernatants with the SBV-strains mentioned above as well as with SIMV, DOUV, SATV, SABOV, SHAV, AKAV, AINOV, and OROV was additionally analyzed by Western blot (WB). The viruses were propagated on BHK-cells (L0164 CCLV) and were deep frozen at −70 °C when a generalized cytopathic effect became evident after 24 – 48 h post infection. Uninfected BHK-cells were used as a negative control. The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions and transferred onto a nitrocellulose membrane using a Trans-Blot SD Semi-Dry Transfer Cell device (Bio-Rad). The nitrocellulose membranes were blocked for 1 h using 5% non-fat dry milk diluted in PBS and subsequently incubated with the hybridoma supernatants (diluted 1:20 in PBS) overnight at 4 °C followed by a horseradish peroxidase-conjugated anti-mouse antibody (Dako, diluted 1:200 in PBS) for 1 h at room temperature. Proteins were visualized using the Super Signal West Pico Chemiluminescent substrat (Thermo Scientific).

The Western blot was performed based on the general protocol. SDS-PAGE with 10% gel was performed and followed by a semidry blotting method using PVDF membrane (Immobilon-P membrane, 0.45 µm) using Bio-Rad Trans-Blot SD Semi-Dry transfer cell device. 5 (m/v)% low-fat milk powder in TTBS was used for blocking, for 1 h at room temperature or overnight at 4 °C. Primary antibodies were applied in 0.5 (m/v)% low-fat milk powder containing TTBS for 1 h at room temperature or overnight at 4 °C. After washing four times for 8 min with TTBS, the secondary antibody was added in TTBS containing 0.5 (m/v)% low-fat milk powder. The following antibodies were used in the study: Monoclonal Transglutaminase-4 antibody (1C6) (Covalab) (1/2500), Polyclonal antibody to human epidermal transglutaminase (TG3) (Zedira) (1/2000), Monoclonal anti-polyHis/HRP antibody (Sigma) (1/20,000), Streptavidin/HRP (1/2500) (BioLegend), Goat-anti-mouse IgG (R-05071-500, Advansta, San Jose, CA, USA) (1/10,000) and Goat-anti-rabbit IgG (H + L), HRP conjugate (R-05072-500, Advansta) (1/10,000).