Amersham hybond p pvdf

Protein samples in Laemmli buffer were resolved by SDS-PAGE, the proteins were transferred on an Amersham Protran western blotting nitrocellulose (GE Healthcare) or an Amersham Hybond P PVDF (GE Healthcare) membrane by semi-dry transfer. The membrane was blocked with 1 × TBS containing 0.1% Tween-20 and 10% skimmed milk, and incubated with primary antibodies (see the Supplementary Table S4 for the complete list of antibodies) diluted in 1× TBS, 0.1% Tween-20 for 1 h at room temperature or overnight at 4°C. The membrane was washed with 1× TBS, 0.1% Tween-20 and incubated with a dilution of the corresponding HRP-coupled secondary antibody for 30 min, followed by another round of washing. The chemiluminescent signal was visualized with the SuperSignal West Pico PLUS (Thermo Fisher Scientific), or Westar Sun (Cyanagen), or ECL Select Western blotting (GE Healthcare) chemiluminescent substrate on ChemiDoc Touch (Bio-Rad) or G-Box (Syngene) and analysed with Image Lab (v. 5.2.1).

Proteins were separated on SDS-PAGE. The gels were either stained with CBB or proteins were transferred onto a PVDF membrane (Amersham Hybond P PVDF, GE Healthcare). The blot was probed with anti-Bet v 1, mouse monoclonal 2E10 (Indoor Biotechnologies, Ltd.), or anti-6x His tag mouse monoclonal antibody (Thermo Fisher Scientific).