Ecl advance reagents

A total of 100-200 mouse oocytes were placed in 5^*SDS sample buffer and heated at 100 °C for 5 min,then cooled on ice and centrifuged at 14000 rpm for 5 min to remove pellets. Proteins were separated by SDS-PAGE with a 4% stacking gel and a 10% separating gel for 1.5 h at 100 voltage (V) and then electrophoretically transferred to polyvinylidene fluoride membranes for 1.5 h at 100 V at 4 °C. After being washed three times (10 min each time) in TBS (20 mM Tris, 137 mM NaCl, pH 7.4),Then membranes were blocked in TBST (TBS with 0.1% Tween 20) containing 5% non-fat milk for 1 h at room temperature, followed by incubation at 4 °C overnight with primary antibody. The next day, after washing three times in TBST (10 min each), membranes were incubated at room temperature for 1 h with horseradish peroxidase-conjugated Pierce anti-mouse IgG (1:5,000) or anti-Rabbit IgG (1:5000), followed by three times washing in TBST(10 min each). Finally signal detection was performed by the enhanced chemiluminescence (ECL) technique using ECL Advance reagents (Amersham Biosciences UK Limited, Little Chalfont Buckinghamshire, England).

100 oocytes were collected in SDS sample buffer (Biorad) and boiled for 4–5 min, then cooled on ice and centrifuged at 14000 rpm for 5 min to remove pellets. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a 4% stacking gel and a 10% separating gel for 1.5 h at 100 voltage (V) and then electrophoretically transferred onto polyvinylidene fluoride (PVDF) membrane for 1.5 h at 100 V at 4°C. After being washed three times (10 min each time) in TBS (20 mM Tris, 137 mM NaCl, pH 7.4), the PVDF membrane was blocked in blocking buffer (TBS with 0.04% Tween20 and 2.5% low-fat milk) at room temperature for 1 h. Next, the membrane was in sequence incubated with goat anti-trim75 antibody diluted 1:500 in blocking buffer at 4°C overnight, washed three times (10 min each) in TBS with 0.04% Tween20 (TBST), incubated with HRP-conjugated Rabbit anti-Goat IgG (H+L) diluted 1:5000 in blocking buffer at room temperature for 1 h, washed three times (10 min each) in TBST. Finally signal detection was performed by the enhanced chemiluminescence (ECL) technique using ECL Advance reagents (Amersham Biosciences UK Limited, Little Chalfont Buckinghamshire, England). For actin detection, the dilution for mouse anti-actin antibody is 1:3000, the dilution for HRP-conjugated goat anti-mouse IgG (H+L) dilution is also 1:5000.