Hrp conjugated goat anti mouse

Ovaries from 30 females were collected and homogenized in lysis buffer (20mM Hepes pH7.9, 100mM KCl, 0.1mM EDTA, 0.1mM EGTA, 5% Glycerol, 0.05% Igepal and protease inhibitors (Roche)). The protein extracts were cleared by centrifugation and stored at -80°C.
Eggs were collected every 30 min, dechorionated in bleach and quickly frozen in liquid nitrogen. Protein extracts were prepared from ca. 10 μl of embryos. Protein samples were run on 15% SDS polyacrylamide gel and transferred to Immun-Blot^® PVDF membrane (Bio-Rad) for 1h at 60V. Membranes were blocked for 1h at room temperature in 5% non-fat milk in PBS 1X-Tween20 0.05%, followed by an overnight incubation with the primary antibody at 4°C in 5% non-fat milk in PBS1X-Tween20 0.05%. Secondary antibodies used were added and incubated for 2 hours at room temperature. Protein detection was performed using ECL solution according manufacturer’s instruction (GE Healthcare). Antibodies used were: rabbit polyclonal anti-DHD (1/1000) [6 (link)], mouse monoclonal anti-α-Tubulin (Sigma Aldrich #T9026, 1:500), HRP-conjugated goat anti-mouse (Biorad #170–5047; 1:50 000) and peroxidase-conjugated goat anti-rabbit (Thermoscientific #32460; 1:20 000).

Western blots were performed as previously described (Eng et al., 2015 (link)). Primary antibodies used were a mouse monoclonal anti-V5 used at a 1:5000 dilution (Invitrogen, Carlsbad, CA) and a mouse monoclonal anti-Tub1 used at 1:20,000 dilution. Secondary antibody used was an HRP-conjugated goat anti-mouse at 1:20,000 (BioRad, Hercules, CA).