4.2. Isolation of Seminal Plasma Protein Fractions

Seminal plasma proteins were separated on an affinity heparin–sepharose column (GE Healthcare, Uppsala, Sweden) into two fractions: heparin-binding (Hep+) and heparin-non-binding (Hep−), which were added after dialysis and lyophilization to the cryopreservation medium at the final three protein concentrations: 125 µg/mL, 250 µg/mL, and 500 µg/mL. The seminal plasma sample was thawed at room temperature and then centrifuged for final purification at 10,000 g for 5 min. The sample was then loaded onto a heparin–sepharose column, which was attached to a pump and an automatic collector. The pump flow rate for the Hep− fraction collection was 1.5 mL/10 min. When collecting the Hep− fraction, the column was aspirated with a PBS buffer (phosphate-buffered saline: 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂HPO₄, pH 7.4) to wash the column after aspirating the seminal plasma. After 150 min, the column was transferred to a 3 M NaCl solution in PBS, at a flow rate of 4 mL/10 min for 60 min, which released bound Hep+ proteins from the column. Separated samples in tubes were measured by a spectrophotometer (Biochrom, Libra S22, Fisher Scientific, Walthman, MA, USA) at 280 nm for protein content. Fractions with an absorbance above 0.03 were frozen and left in the freezer before protein fraction preparation. The individual protein fractions were subsequently dialyzed and lyophilized. Dialysis was done through membrane-cell dialysis tubing (MWCO 3500, SERVA, Heidelberg, Germany) for 48 h in a 0.2% acetic acid solution [80]. The acetic acid solution was exchanged at least three times over two days to remove all salts from the protein fraction solution. After dialysis, the fraction solutions were measured on a conductometer (Eutech ECTestr 11, OAKTON Instruments, Vernon Hills, IL, USA) to determine the salt content and dialysis efficiency. Before subsequent lyophilization, samples of protein fractions were placed in a freezer in lyophilization flasks. The frozen protein fractions were lyophilized (LYOVAC GT 2 E, FINN-AQUA) overnight until dry.