Perfectblue electro blotter

The recombinant proteins were separated by denaturing SDS-PAGE in 12.5% gels, then transferred onto nitrocellulose membranes by semi-dry blotting at 0.8 mA/cm² for 1 h (PerfectBlueTM Electro Blotter, Peqlab, Erlangen, Germany). Membranes were blocked with 5% skim milk powder in Tris-buffered saline (TBS) supplemented with 0.05% (v/v) Tween 20 (TBST) for 1 h. Specific mAbs or sera were appropriately diluted in TBST and incubated on the membranes for one hour at room temperature. The membranes were washed three times with TBST and incubated for another hour with anti-murine IgG or gallid IgY HRPO-labeled secondary antibody conjugate (Santa Cruz Biotechnology, Heidelberg, Germany). Blots were washed three times and incubated with SuperSignal™ West Pico Chemiluminescent substrate solution (ThermoScientific, Braunschweig, Germany) before being analyzed on an imaging system (VersaDoc, Bio-Rad, Munich, Germany). Photos were edited with respect to contrast and brightness and composite blots were assembled using cut-out lanes (GIMP software, version 2.10.32).

Protein samples were separated by SDS-PAGE in 12.5% gels. For denaturing conditions samples were heated in Laemmli loading buffer containing dithioerythritol (DTE). Proteins were also separated under non-denaturing conditions in 10% PAGE without SDS and DTE. Proteins were transferred onto nitrocellulose membranes by semi-dry blotting at 0.8 mA/cm² for 1 h (PerfectBlueTM Electro Blotter, Peqlab, Erlangen, Germany). Membranes were blocked with 5% skim milk powder in Tris-buffered saline (TBS) supplemented with 0.05% (v/v) Tween (TBST) for 1 h. Specific antibodies and sera were appropriately diluted in TBST and incubated on the membranes for 1 h at room temperature. Membranes were washed three times with TBST and incubated for another hour with POD-labeled secondary antibody. Blots were washed again for three times, then incubated with substrate SuperSignal™ West Pico Chemiluminescent substrate solution (ThermoScientific, Braunschweig, Germany) before being analyzed with an imaging system (VersaDoc, Bio-Rad). Photos were edited with respect to contrast and brightness and composite blots were assembled using cut-out lanes (GIMP software).