Secondary horse radish peroxidase coupled antibodies

Cells were harvested in ice-cold phosphate-buffered saline (PBS). Harvested cells were lysed in RIPA buffer (Cell signaling) supplemented with complete protease and phosphatase inhibitors (PIM complete; Roche) for 20 min on ice. After centrifugation at 15,000 g for 15 min at 4 °C, total protein in whole-cell extracts was quantified using Rotiquant (Carl Roth). In all, 40 µg of protein was resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Invitrogen) and blotted on polyvinylidene fluoride membranes (Invitrogen). The membranes were probed with anti-OCT6 or anti-β actin (Santa Cruz) primary antibodies followed by secondary horse-radish peroxidase-coupled antibodies (Rockland Immunochemicals), and signals were acquired in a chemiluminescence detection system (Applied Biosystems) in a linear dynamic range.

All reagents, buffers, and devices were supplied by ThermoFisher Scientific unless otherwise stated. 30µl of PBS containing native or gas plasma‐treated Ova (30 µg for coomassie, 15 µg for western blot) were mixed with 4x NuPAGE LDS sample buffer and loaded without denaturation on a 10‐well 4–12% Bis‐Tris Gel. SeeBlue prestained standard was loaded, and gel electrophoresis was performed in a chamber filled with 1x MES SDS running buffer and connected to a power supply (Biometra Analytik‐Jena, Germany). For coomassie, gels were stained with 4% coomassie brilliant blue R250 in 80% methanol, 20% acetic acid (both Carl Roth, Germany), and washed with a de‐staining solution (20% methanol, 10% acetic acid, 70% ddH₂O). For western blot, proteins were blotted on an activated PDVF membrane, blocked with Rotifix (Carl Roth, Germany), and stained with anti‐Ova polyclonal primary antibody (Biozol, Germany) followed by secondary horse‐radish peroxidase‐coupled antibodies (Rockland Immunochemicals). Signals were acquired after adding ECL reagent super signal WestPicoPlus in a chemiluminescence detection system (GE Healthcare, USA).