Molecular weight analysis by gel permeation chromatography (GPC)

The water diluted polysaccharide samples (~8 μg/μl) have been analyzed for molecular weight. Six dextran GPC analytical standards (Sigma-Aldrich^®) in the range of 25 kDa to 1,400 kDa have been used for molecular weight calibration. The samples and the standards have been filtered through 0.2 μm filters and then analyzed by GPC using Refrectomax 520 RI (Thermo Fisher Scientific ^™) detector and HPLC Finnigan Surveyor System (Thermo Fisher Scientific ^™) controlled by Chromeleon ^™ v.7 software (Thermo Scientific ^™). The separation of MWs has been performed by Superose ^™ 6 10/300 column (GE Healthcare ^™), using water mobile phase at the flow rate of 0.2 ml/min (back pressures 277 psi) over a period of 120 minutes. The Superose ^™ 6 10/300 column is designed for the fractionation range of M_p * 1000 to 3 × 10⁵ (dextrans) which covers the range of analyzed XyG [49]. In order to determine whether there was a difference between genetically modified and control root samples, GPC spectra have been compared by Principal Component Analysis (PCA) using the Origin 2018 software (version 95E).