G3000pwxl

The molar mass distribution of the liquid samples obtained by autohydrolysis was studied at least in duplicate. Analyses were performed in an Agilent 1100 equipment, with two columns in series, 300 × 7.8 mm TSKGel G2500PW_XL and G3000PW_XL from Tosoh Bioscience (Griesheim, Germany) and a PW_X-guard column (40 × 6 mm). The operation conditions were: 70 °C using Milli-Q water at 0.4 mL/min as mobile phase. To evaluate the molar mass distribution, dextrans (1000–80,000 g/mol) (Fluka, St. Gallen, Switzerland were used as standards.

The murine lenses were homogenized, and water-soluble proteins were extracted as described previously [10 (link)]. The molecular weights of the extracted proteins were determined from the light scatter data, and the concentrations were calculated based on the refractive index data. In the studies involving water-soluble proteins from WT and knock-in mouse lenses, 100 μl of water-soluble lens proteins were separated in succession on G3000 PWXL and G5000 PWXL size exclusion chromatography columns (Tosoh Bioscience LLC, Prussia, PA) in line with the Viscotek TDA 302 triple detector array system, which measured UV absorption, refractive index (RI), multi-angle light scattering, and viscosity (Viscotek/Malvern). The GPC system was equipped with a VE-1122 pump and a VE-7510 degasser. The Viscotek OmniSEC software was used to calculate the molecular weight of the crystallin proteins using bovine serum albumin (BSA) and the 92-kDa Pullulan Malvern standards. The protein samples (100 μl) were injected onto the columns, and Dulbecco’s Phosphate-Buffered Saline (PBS) Modified buffer (0.5x) without Ca²⁺ or Mg²⁺ was used as the mobile phase at a flow rate of 0.8 ml/min at 25°C. The protein concentration was calculated based on the refractive index using a dn/dc of 0.185 for both the BSA standard and the crystallin proteins.

HPSEC (LC-20 AD, Shimadzu, Kyoto, Japan) equipped with a PDA detector (SPD-M20A, Shimadzu, Kyoto, Japan) and RID-10 detector (Shimadzu, Kyoto, Japan) with a TSK column (G3000PWXL, TOSOH, Tokyo, Japan) was used to measure the molecular weight distribution in EPS. Column temperature was maintained at 40 °C and it was calibrated using polyethylene glycol (PEG) as a standard. Milli-Q water was used as the mobile phase at a flow rate of 0.5 mL min⁻¹ and injection volume was 100 mL. In HPSEC chromatographs, UV254 signals intensity aims to detect protein content shown in red color line, while black line shows RID signals intensity, representing the polysaccharide components in EPS. The principle of HPSEC based on difference in retention times/intensity of the molecules which is characterized by size and concentration. Small molecules can penetrate the column of HPSEC chromatography quickly, while very large molecules penetrate the column slowly [24 (link)].