Immunoblotting of Basic Sperm Proteins

For immunoblotting of AU-PAGE, an equivalent of 1.5M sperm of pooled basic protein extraction was used. The proteins were transferred towards the negative pole onto a PVDF membrane (pore size 0.45μm; Roth, Karlsruhe Germany), blocked and incubated with the primary antibody overnight (antibody dilutions shown in S3 Table). For SDS PAGE, proteins were transferred towards the positive pole onto a PVDF membrane (pore size 0.45μm; Roth, Karlsruhe Germany). The membranes were then washed and incubated with an HRP-labelled secondary antibody followed by chemiluminescent detection using Westar NOVA 2.0 chemiluminescent substrate (Cyanagen, Bologna, Italy) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, USA). Since Merges et al. [38 (link)] was able to show that (outer dense fiber protein 2) ODF2, which is a major component of the sperm tail, is compatible with AU-PAGE and represents one of the prominent bands in the upper part of the Coomassie AU gel we used the protein as a loading control.