Horseradish peroxidase conjugated goat anti rabbit

To determine levels of claudin-4 protein expression, tumor cells were scraped from culture plates in presence of lysis buffer (30 mM Tris HCl pH7.4, 150 mM NaCl, 1% TritonX-100, 10% glycerol, 2 mM EDTA, 0.57 mM PMSF, 1X cOmplete™ Protease Inhibitor Cocktail), placed on a shaker for 10 minutes and spun at 13,000 rpm for 10 minutes. Supernatant was collected and 20 ug of total protein was denatured, resolved on 10% SDSPAGE, and transferred to a polyvinylidene difluoride (PVDF) membrane (Bio Rad, Hercules, CA, USA). Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for one hour at room temperature (RT) before treatment with either rabbit anti-human claudin-4 (1:500, Invitrogen), mouse anti-human claudin-4 (1:500; Invitrogen), or rabbit anti-human GAPDH (1:10,000; Sigma) overnight at 4°C. Membranes were then washed 4 times with TBST for 15 minutes before treatment with horseradish peroxidase-conjugated goat anti-rabbit (1:10,000; GE Healthcare, Buckinghamshire, UK) or goat anti-mouse (1:10,000; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) antibodies for 1 hour at room temperature. Membranes were washed with TBST as described above and then visualized using an ECL Prime Western Blotting Detection Reagent (GE Healthcare) and X-ray film (CLXPosure Film, Thermo Scientific, Rockford, IL, USA).