Biometra flashblot

Protein samples separated on SDS-PAGE gels were transferred to the polyvinylidene fluoride (PVDF) membrane. The semi-dry transfer was performed in Tris-glycine buffer with SDS and isopropanol using Biometra Flashblot (Analytik Jena GmbH). Parameters of the analysis were set to 15 V, 250 mA for 45 min. Subsequently, the PVDF membrane was incubated in specific antibody solution (mouse anti-His) (Invitrogen) with dilution of 1:1000 in a 1% skim milk solution at 4 °C overnight. Further, the membrane was washed in goat anti-mouse secondary antibody (1:10 000) in 1% skim milk solution (Goat Anti-Mouse IgG, Sigma Aldrich) for 1 h at room temperature. For signal visualisation, the membrane was washed in chemiluminescence solution using Clarity Max Western ECL Substrate kit (Bio-Rad) according to the protocol and signal was detected using ImageQuant LAS 500 chemiluminescence CCD camera (GE Healthcare).

Protein samples separated on SDS-PAGE gels were transferred to the polyvinylidene fluoride (PVDF) membrane. The semi-dry transfer was performed in Tris-glycine buffer with SDS and isopropanol using Biometra Flashblot (Analytik Jena GmbH). Parameters of the analysis were set to 15 V, 250 mA for 45 min. Subsequently, the PVDF membrane was incubated in specific antibody solution (mouse anti-His) (Invitrogen) with dilution of 1:1000 in a 1% skim milk solution at 4 °C overnight. Further, the membrane was washed in goat anti-mouse secondary antibody (1:10 000) in 1% skim milk solution (Goat Anti-Mouse IgG, Sigma Aldrich) for 1 h at room temperature. For signal visualisation, the membrane was washed in chemiluminescence solution using Clarity Max Western ECL Substrate kit (Bio-Rad) according to the protocol and signal was detected using ImageQuant LAS 500 chemiluminescence CCD camera (GE Healthcare).