Donkey anti sheep hrp

1 x 10 7 cells lysed in NuPAGE sample buffer (supplemented with β-mercaptoethanol) were loaded on to a 4-12% NuPAGE Bis Tris Gel and run at 150V in MOPS buffer. The gels were transferred onto a PVDF membrane using the wet transfer system XCell II Blot Module of Invitrogen at a constant voltage of 30V for 90 min. The blot was blocked with 5% Skimmed Milk (Sigma) for 1 h. Primary antibodies were added at the following concentrations: rabbit polyclonal PNT1 antibody-1:500 overnight at 4°C, sheep polyclonal OPB antibody-1:20,000 1 h at room temperature. After 3 washes in 1X TBST, goat anti rabbit HRP (Promega) and donkey anti-sheep HRP (Santa Cruz) were added at 1;5000 dilution for 1 h. The blots were washed with 1X TBST and overlayed with Clarity Max substrate (BioRad). The blots were developed in a myECL Imager (Thermo Fisher Scientific).