Nativepage 20x cathode additive supplemented native running buffer

HEK 293T cell supernatants were diluted in NativePAGE sample buffer with 5% G-250 sample additive (Invitrogen) and loaded in a NativePAGE 4–16% Bis-Tris gel (Invitrogen). Gel electrophoresis was carried out at 150V in Dark Blue Buffer (5% NativePAGE 20X Cathode Additive supplemented native running buffer, Invitrogen) until the dye front migration reached 1/3 of the gel. Gel electrophoresis was continued in Light Blue Buffer (0.5% NativePAGE 20X Cathode Additive supplemented native running buffer, Invitrogen) until protein migration reached the end of the gel. After gel electrophoresis, proteins were transferred to a PVDF membrane by western blotting at 25V for 1 hour. After transfer, the proteins were fixed by incubating the membrane with 8% acetic acid for 15 minutes. The membrane was rinsed with deionized water, air-dried and incubated with ethanol until dye was removed. The membrane was incubated in blocking buffer (PBS 1% bovine serum albumin 0.2% skim milk powder) for 30 minutes. The proteins were probed using E2-specific AR3A antibody [30 (link)] and HRP-conjugated F(ab’)2-goat anti-human IgG Fc secondary antibody (Invitrogen) and visualized by chemiluminescence.