Goat anti mouse secondary antibody conjugated to horseradish peroxidase

Protein levels of TEX101, LY6K, ADAM29, and DPEP3 were assessed by Western blot analysis. Twenty μg of total protein from one rs35033974 homozygous and one WT spermatozoa lysate were loaded onto an SDS-PAGE gel (4–15%, Bio-Rad) and transferred onto PVDF membranes (Bio-Rad). After blocking, membranes were incubated overnight at 4 °C with rabbit polyclonal antibodies against TEX101 and DPEP3 (HPA041915 and HPA058607, Sigma-Aldrich, St. Louis, MO), sheep polyclonal antibody against LY6K (AF6648, R&D Systems, Minneapolis, MN), mouse monoclonal antibody against ADAM29 (H00011086-M09, Abnova Corporation, Walnut, CA), and GAPDH antibody (AM4300, Thermo Fisher Scientific, Rockford, IL). The membranes were washed then incubated with goat anti-rabbit, donkey anti-sheep, and goat anti-mouse secondary antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA). Proteins were detected with chemiluminescence substrate (GE Healthcare Life Sciences, Mississauga, ON).

Whole mouse brains were quickly frozen on dry ice and stored at − 80 °C before extraction. Tissue from eight mice were thawed, individually sonicated in 4% SDS/50 mM Tris, pH 7.6, and heated for 10 min at 90 °C. Protein concentrations for all fractions were determined using the BCA assay (Pierce, Waltham, MA, USA), using bovine serum albumin as a standard. Samples were normalized for total protein content and SDS-containing sample buffer was added to samples, which were then further heated for 10 min at 90 °C. Protein samples were separated on SDS-polyacrylamide gels (8% or 15%) and transferred electrophoretically onto 0.22 μm nitrocellulose membranes (Bio-Rad, Hercules, CA). Membranes were blocked in 5% non-fat milk in Tris-buffered saline, pH 7.6 (TBS) for 1 h at room temperature, then incubated in primary antibodies (detailed in Table 2) diluted in 5% non-fat milk/TBS block solution overnight in 4 °C. After incubation, membranes were rinsed with agitation in TBS for 5 min, repeated eight times. Membranes were then incubated with goat anti-mouse secondary antibody conjugated to horseradish peroxidase (Jackson Immuno Research Labs, Westgrove, PA), diluted 1:1000 in 5% non-fat milk/TBS for 2 h at room temperature. Protein band signal was detected with Western Lightning-Plus ECL reagents (PerkinElmer, Waltham, MA) and chemiluminescence imaging (PXi, Syngene, Frederick, MD).