2.3 Western blotting
Proteins were extracted from sperm cells. Protein quantitation was
performed by BCA Protein Assay (23227, Thermo Fisher) according to the
manufacturer’s instructions. Next, the proteins were denatured at 95°C
for 10 min. The denatured proteins were separated on 10%
SDS-polyacrylamide gels (stock gel: 60 V, 30 min; separating gel: 120 V,
1.0 h) and then transferred into a 0.45 µm pore size polyvinylidene
difluoride (PVDF) membrane (ISEQ, 00010, Millipore) by wet transfer. The
transferred membrane was blocked with 5% skimmed milk for 1 h at room
temperature and incubated in primary antibody: anti-CFAP47 (1:500,
sc-514714, Santa Cruz Biotechnology); anti-GAPDH (1:1000, ab8245, Abcam)
solution at 4°C overnight. Subsequently, the membrane was washed with 1
× TBST three times, and each time, the membrane was cleaned for 5 min.
Then, the membrane was incubated with goat anti-mouse IgG secondary
antibody-HRP (1:5000, 32230, Thermo Fisher Scientific) in 5% skimmed
milk at room temperature for 1.5 h, and the next step was to wash the
membrane with 1 × TBST three times. The time for cleaning the membrane
was the same as above. Finally, immunoblots were developed using Thermo
Scientific™ Pierce™ ECL Western Blotting Substrate (TWBKLS0500,
Millipore).