5.8. Immunoprecipitation and Western Blotting of Acetylated Proteins

Acetylation of sperm proteins in both unilateral (n = 6) and bilateral varicocele patients (n = 6) was demonstrated using WB. Immunoprecipitation of acetylated proteins was carried out using anti-acetyl Lysine antibody (ab190479, Abcam, USA) followed by WB detection of selected acetylated proteins. The criteria applied for the selection of DEPs involved in the acetylation process were as follows: (i) Proteins involved in the networks; (ii) abundance of the protein must be moderate or high in any one group; and (iii) proteins with a well-described function in the literature. Four proteins (ANXA2, HIST1H2BA, SERPINB6, and SOD1) were chosen for validation by WB in both the unilateral and bilateral varicocele group.

Immunoprecipitated acetylated proteins were first loaded into a 4–15% SDS–PAGE for 2 h at 90 V. The resolved proteins were transferred onto polyvinylidene difluoride (PVDF) membranes and analyzed as described earlier [52]. The expression levels of the WB-validated proteins were normalized against the global acetylated proteins (Supplementary Figure S1) and compared between unilateral and bilateral varicocele using the Mann–Whitney test and p < 0.05 was considered significant. Data analysis was performed using MedCalc Statistical Software (version 17.8; MedCalc Software, Ostend, Belgium).