Tskgel supersw3000

The changes in molecular weight of the protein fortifiers with time were analyzed using a chromatographic system Hitachi (Merck) with a UV detector and a data station. The system was equipped with a SEC-HPLC column TSKgel SuperSW3000 (TOSOH) with dimensions 300 × 4.5 mm and a guard column 3 cm × 4.5 mm, the bed particle diameter was 4 μm. The measurement conditions were: phosphate buffer pH 7 as the eluent, the flow rate 0.2 mL min^− 1, the temperature for the measurement 22 °C, the injection volume 20 μL. Chromatograms were recorded by the detector at wavelength 280 nm.

The monitoring of anti-procollagen II antibody immobilization and cross-linking was made by SEC-UV analysis of supernatants. The analyses were carried out using a TSKgel SuperSW3000 (4.6 mm ID × 300 mm L, 4 μm) from Tosoh Bioscience GmbH (Griesheim, Germany). Analytical conditions were set according to the producer’s recommendations: mobile phase 0.1 M Na₂SO₄, 0.05% NaN₃ in 0.1 M sodium phosphate buffer, pH 6.7; flow rate 0.35 mL/min; and column temperature maintained at 25 °C. The injection volume was 5 μL and UV detection was carried out at 280 nm.

The molecular weights of SF degummed for different times were estimated by SEC-UV analysis. Chromatographic separations were performed on an Agilent HPLC series 1200 system (Santa Clara, CA, USA), equipped with mobile phase online degasser, quaternary pump, autosampler, thermostated column compartment, and diode array detector. For data acquisition, the ChemStation software version Rev. B.04.01 (Agilent Technologies, Santa Clara, CA, USA) was used. The analytical method, previously reported [48 (link)], entails the use of a TSKgel SuperSW3000 (4.6 × 300 mm; Tosoh Bioscience, Tokyo, Japan) and a mobile phase composed of 0.1 M Na₂SO₄ and 0.05% (w/v) NaN₃ in 0.1 M phosphate buffer, pH 6.7. Flow rate, column temperature, and injection volume were set at 350 μL/min, 25 °C, and 5 μL, respectively. UV absorption was monitored at 280 nm. For MW estimation, a calibration curve was constructed using protein standards (ribonuclease A, 14 kDa; β-lactoglobulin (dimeric form), 37 kDa; bovine serum albumin, 67 kDa; rituximab, 145 kDa; thyroglobulin, 660 kDa; y = −0.9949 x + 4.9497; R² = 0.9934). SF samples were diluted with water to a final concentration of 0.33% w/v before injection, and analyzed in triplicate.

All analyses were performed on an Agilent 1200 HPLC (Agilent, Santa Clara, CA) using a TSKgel SuperSW3000 2.0 mm × 30 cm, 4 µm column (Tosoh Biosciences Tokyo, Japan) isocratically in 50 mM sodium phosphate pH 7.0 containing 250 mM NaCl at a flow rate 75 µl/min for 30 min. Injection volumes were 3 µl at a protein concentration of 150 µg/ml. Protein molecular weight standards were purchased from Sigma (#69385) and reconstituted according to the manufacturer’s directions. Fluorescence detection was monitored at 295 nm excitation and 345 nm emission. Data analysis was performed using ChemStation software (Agilent).