Purification of Protamine by Sephadex G50

A modified version of the method proposed by Aida Karray et al. [76 (link)] was used. An appropriate amount of sephadex G50 was weighed and swelled in pure water at a ratio of 1:10. The swelling process included incubation at 20 °C and 90 °C for 3 h and 1 h, respectively. After washing with water 2–3 times and discarding the supernatant, the swollen sephadex G50 was packed into a column (Φ1.00 cm × 100.00 cm), and it settled naturally. The packed gel column was free of bubbles and layers and exhibited a uniform appearance. The detection wavelength was set to 215 nm, and the flow rate was maintained at 1 mL/min with a column pressure below 1 MPa. The column was equilibrated with a pH of 5.4 and a 25 mmol/L acetic acid–sodium acetate buffer using 2–3 column volumes (CVs). Once the baseline stabilized, the protamine solution was filtered using a 0.45 μm microporous membrane before injection, with a sample volume of 5 mL per run. Each fraction of 4 mL was collected, and the UV detection peaks were subjected to a Bradford assay. Fractions containing the same components were combined, concentrated, and stored at −80 °C for future use.