Sperm Protein Phosphorylation Profiling

Unless otherwise stated, chemicals and reagents were purchased from Sigma-Aldrich Quimica S.A. (Madrid, Spain). Sydney IVF Sperm Medium was provided by Cook Medical (Barcelona, Spain). 4–15% polyacrylamide gels were supplied by Bio-Rad (Madrid, Spain) and PVDF membranes by Merck (Madrid, Spain). The primary antibodies were: anti-phospho-PKA substrates (PKAs-P) (Cell Signaling Technology, Beverly, USA, #9624), anti-phosphotyrosine (Tyr-P) (Abcam, Cambridge, UK, #ab10321) and anti-β-tubulin (β-TUB) (Sigma-Aldrich Quimica S.A., Madrid, Spain, #T0198)^{78 (link),79 (link)}. Horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgGs were obtained from Santa Cruz Biotechnology (Heidelberg, Germany, #sc2004) and Bio-Rad (Madrid, Spain, #1706516), respectively. PageBlue was provided by Thermo Scientific (Rockford, IL, USA), whereas acetonitrile, trifluoroacetic acid and formic acid by Fisher Scientific (UK). Trypsin Gold Proteomics Grade and ProteaseMax surfactant were purchased from Promega Corporation (Madison, MI, USA).