4.5. Acid-Urea Polyacrylamide Gel Electrophoresis

Human protamines were analyzed by AU-PAGE as previously described [17]. In brief, the components of gel were 15 mL of a solution composed by 2.5 M urea, 0.9 M acetic acid, and 15% acrylamide/0.1% N,N’-Methylene-bis-acrylamide, 80 µL of TEMED and 800 µL of 10% APS. After gelification in about 1 h, at room temperature, pre-electrophoresis was performed at 150 V for 1 h 30 min, placing the negative electrode at the bottom of the gel. The buffer used was 0.9 N acetic acid and in the wells were loaded 20 µL of a solution containing 0.9 N acetic acid 2.5 molar urea. After pre-electrophoresis, the wells were washed with 0.9 N acetic acid buffer using a syringe, and the electrophoretic chamber was filled with fresh 0.9 N acetic acid buffer. Then, 2–2.5 µL per well of each sample, containing 4 µg of proteins in 0.9 N acetic acid and 2.5 molar urea, were loaded for the run, which was conducted at 150 V for about 55 min. At the end of the electrophoresis, gels were stained with Amido Black, and then with Coumassie Blue Brilliant R-250, as previously described [60]. Gels were acquired using a Gel-Doc system (BioRad, Hercules, CA, USA) through Quantity One v.4.4.0 (BioRad, Hercules, CA, USA) software. A densitometric analysis of the bands on the gel was performed using the software ImageJ ver 1.50d (Wayne Rasband, National Institute of Health, Bethesda, ML, USA, https://imagej.nih.gov/ij/, 1997–2018).