Peroxide conjugated secondary antibody

After removal of the surrounding granulosa cells, denuded GV and MII oocytes were frozen in RIPA buffer (1M Tris-HCl pH 7.4, 5 M NaCl, 5% sodium deoxycholate, 10% SDS, 10% Nonidet P40, 0.5 M EGTA) supplemented with a protease and phosphatase inhibitor solution (Thermo Scientific, Waltham, MA). Prior to analysis the samples were thawed on ice, mixed with 5x loading buffer, and heated at 98°C for 7 min. The proteins were separated in 8% acrylamide gels containing 10% SDS, then transferred onto hydrophobic PVDF membranes (Millipore). Following transfer, the membrane was blocked in Tris Buffered Saline supplemented with 2% Tween-20 (TBST) and 5% milk powder, then incubated with anti-CEP215 (Millipore, 1/1000 dilution) at 4°C overnight and subsequently washed in TBST. A peroxide-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA) was added for 1h and the membrane then washed in TBST. An ECL kit (Millipore) was used for chemiluminescent detection. The membrane was also probed with anti-β tubulin (Sigma Aldrich, 1/2000 dilution) as a control, under similar conditions. Individual band intensity was quantified, for three independent experimental replicates, using the Image J software and the relative total protein values in each group were compared to the control, which was normalized to 1.0.

Oocytes (n=50 per group) were frozen in RIPA buffer supplemented with a protease and phosphatase inhibitor solution. The samples were thawed on ice and mixed with 5x loading buffer, then heated at 98°C for 7 min. Proteins were separated in 10% acrylamide gels, then transferred onto a hydrophobic PVDF membranes (Milipore, Burlington, MA). The membrane was blocked in TBST supplemented with 2% Tween-20 and 5% FBS for 1h at room temperature, and incubated with anti-pT288 AURKA (1/1000; Cell Signaling Technology, Beverly, MA) at 4°C overnight. The following day, the membrane was washed in TBST and incubated with a peroxide-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA) for 1h. ECL (Millipore) was used for chemiluminescent detection. The membrane was also probed with anti-AURKA (1/1000; Novus Biologicals) and anti-β tubulin (1/2000; Sigma Aldrich) as an internal control, under similar conditions. Individual band intensity was quantified using the ImageJ software and the relative total protein values in each group were compared to the control, which was normalized to 1.0.