2.9.1. Size Exclusion–High–Performance Liquid Chromatography (SE–HPLC)

Protein extraction for SE–HPLC was carried out as follows: freeze-dried paste samples (100 mg) were suspended in 1 mL 100 mM sodium phosphate buffer (pH 7), vortexed for 1 min and mixed in a rotator for 30 min at room temperature. The samples were centrifuged (MPW 350R, MPW Med. Instruments, Warsaw, Poland) at 9000× g for 10 min.

SE–HPLC procedure was performed according to Sęczyk et al. [18]. Samples were analyzed with a Varian ProStar HPLC system (Varian, Palo Alto, CA, USA) equipped with a ProStar diode array detector (DAD). The analytical column was a 600 mm × 7.5 mm COSMOSIL 5-Diol-300-II Packed Column (Nacalai Tesque, Kyoto, Japan). The injection volume of the sample was 50 μL. The proteins were eluted isocratically at 30 °C using a PBS buffer (pH 7.4) with a flow rate of 1 mL/min. The acquisition wavelength was set at 280 nm.