Anti mouse hrp linked secondary antibody

Cells were rinsed with cold PBS and scraped on ice with RIPA buffer (Fisher Scientific) with addition of protease and phosphatase inhibitors (cOmplete ULTRA Tablets and PhosSTOP Phosphatase Inhibitor Cocktail Tablets, Roche). Equal amounts of total protein (20 μg) were separated on 10% polyacrylamide gels for 1.5 h at 100 V in 4°C. Proteins were transferred onto PVDF membranes using a semi-dry transfer for 30 min at 20 V. Membranes were blocked in 5% non-fat milk and incubated overnight with anti-GNRHR primary antibody (1:500; GNRHR03, MA5-11538, Invitrogen) at 4°C. Next day, membranes were washed in TBST and anti-mouse HRP-linked secondary antibody (Abcam) was applied for 30 min at RT. Amersham Biosciences ECL detection system (GE Healthcare, Little Chalfont, UK) was used for signal visualization. Images were taken using ChemiDoc MP imager (Bio-Rad). After visualization membranes were stripped using ReBlot Plus Strong Antibody Stripping Solution (Sigma-Aldrich), blocked in 5% non-fat dry milk and incubated overnight (4°C with gentle agitation) with primary anti-actin beta (ACTB) antibody (1:1000; A2228, Sigma-Aldrich). Intensity of the signals was calculated using ImageJ (NIH) software based on the measurements of three membranes.