Veriblot anti rabbit hrp

Mouse testes were collected then homogenized in lysis buffer (10 mM Tris-HCl pH 7.0, 1 mM EGTA, 1 mM EDTA 10mM benzamidine, 1% NP-40) containing 0.1% BME, 1% PMSF and 1% protease inhibitor cocktail. Lysates corresponding to 350 μm total protein were centrifuged twice at 14,000 xg at 4°C for 30 minutes each. Lysates were precleared with 30 μl of Protein G Sepharose beads (BioVision; Milpitas, CA) by rotating for 1 hour at 4°C. The clarified lysates were then collected following centrifugation and complexes formed by rotating the supernatant overnight at 4°C with 2 μg of AURKA antibody (pA5-32035, Thermo-Fisher Scientific; Waltham, MA). Immune complexes were captured by rotating the sample in 30 μl of beads for 1 hour at 4°C. Beads were washed five times in lysis buffer, denatured by boiling in 2X sample buffer for 5 min and proteins analyzed by SDS-PAGE followed by blotting with rabbit anti-total AURKA (1:250 pA5-32035, Thermo-Fisher Scientific; Waltham, MA) and Veriblot anti-rabbit HRP (1:2500; Abcam; Cambridge, MA). The negative control was precleared lysate without antibody.