Semidry blotting device

To obtain cytosolic cell fractions, oocytes and eggs were homogenized by pipetting in 5x fold volume of OR-2 buffer containing protease inhibitors APMSF and leupeptin, and then centrifuged at 10,000 rpm, 4 °C, for 10 min. The supernatant fractions were collected after centrifugation and heated at 95 °C for 5 min in the presence of SDS-sample buffer. Protein samples were separated by SDS PAGE using 10% polyacrylamide gels and transferred to PVDF membranes using a semidry blotting device (BioRad). The membranes were blocked with T–TBS buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) containing 3 mg/mL bovine serum albumin. To monitor MAPK activation status, the membranes were incubated at RT for 2 h with 100-fold diluted anti-MAPK or with 200-fold diluted anti-phospho MAPK antibodies. After washing, the membranes were treated with 1000-fold diluted biotinylated anti-rabbit IgG, then with the peroxidase-conjugated Streptavidin Biotin Complex Peroxidase Kit reagent, according to the manufacturer’s manual. The immune complexes were detected by color development catalyzed by peroxidase.

To monitor cyclin B2 content and MAPK phosphorylation status, crude cytosolic fractions of oocytes and eggs were heated at 95 °C for 5 min in the presence of SDS–sample buffer (62.5 mM Tris–HCl, pH 6.8, 2% SDS, 10% sucrose, 0.01% BPB, 100 mM DTT). Protein samples were separated by SDS PAGE using 10% polyacrylamide gels and transferred to PVDF membranes using a semidry blotting device from BioRad (Hercules, CA, USA). The membranes were blocked with T–TBS buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) containing 3 mg/mL bovine serum albumin and incubated at room temperature for 2 h with the 200-fold diluted anti-cyclin B2 antibody, or 100-fold diluted anti-phospho MAPK, or 200-fold diluted anti-MAPK antibodies. The membranes were extensively washed with T-TBS buffer and treated with 1000-fold-diluted biotinylated anti-rabbit IgG, then with peroxidase-conjugated streptavidin, as per the manufacturer’s manual for the Streptavidin Biotin Complex Peroxidase Kit. The immune complexes were detected by color development catalyzed by peroxidase in the presence of hydrogen peroxide and diaminobenzidine tetrahydrochloride.

To monitor cyclin B2 contents, crude cytosolic fractions of oocytes and eggs were heated at 95 °C for 5 min in the presence of SDS-sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% sucrose, 0.01% BPB, 100 mM DTT). Protein samples were separated by SDS PAGE using 10% polyacrylamide gels and transferred to PVDF membranes using a semidry blotting device from BioRad (Hercules, CA, USA). Membranes were blocked with T–TBS buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) containing 3 mg/mL bovine serum albumin and incubated at room temperature for 2 h with a 200-fold diluted anti-cyclin B2 antibody. After washing with T-TBS buffer, the membranes were treated with the 1000-fold diluted biotinylated anti-rabbit IgG, then with the peroxidase-conjugated streptavidin, according to the manufacturer’s manual for the Streptavidin Biotin Complex Peroxidase Kit. The immune complexes were detected by color development catalyzed by peroxidase in the presence of hydrogen peroxide and diaminobenzidine tetrahydrochloride.