Endogenous and Recombinant ERK1 Expression in Mouse Eggs

To assess for the presence of endogenous and recombinant expression of ERK1, mouse eggs were collected in SDS buffer, heated for 5 min at 100°C and proteins were separated by SDS-PAGE (Nomikos et al., 2011 (link)). Immunoblotting was then performed as described previously (Verlhac et al., 1996 (link)). Following transfer onto polyvinylidene difluoride membrane (Immobilon-P; Millipore) using a semi-dry transfer system (Trans-Blot SD; Bio-Rad) in buffer (48 mM Tris-HCl, 39 mM glycine, 0.0375% SDS) at 22 V for 4 h and blocking overnight in 5% skimmed (low-fat) milk in TBS (10 mM Tris-HCl, pH 7.5, 140 mM NaCl) containing 0.1% Tween-20 (TBS/Tween), the membrane was incubated for 1 h with the appropriate primary antibody. ERK1 was detected using monoclonal antibody against diphosphorylated ERK1/2 (1∶1000, M9692 Sigma) and ERK1 (1∶500, G-8; sc-271269, Santa Cruz Biotechnology) antibodies. Detection of horseradish-peroxidase-coupled secondary antibody was achieved using enhanced chemiluminescence detection (ECL, Amersham Biosciences). All experiments were repeated at least three times.