λ dna molecular weight marker 3 0.12 21 2 kbp

High Performance Liquid Chromatography-Size Exclusion Chromatography (HPLC-SEC) analysis was used to analyze the size distribution of exopolysaccharide populations and to isolate fractions of different molecular weight. Acid-cleaved exopolysaccharide samples were run on a TSK gel G3000 PWXL column (30 cm X 7.8 mm; particle size 7 μm; cat.# 808021) with a TSK gel PWXL guard column (4.0 cm X 6.0 mm; particle size 12 μm; cat.# 808033) (Tosoh Bioscience). The mobile phase was 0.1 M NaCl, 0.1 M NaH₂PO₄, 5% CH₃CN, pH 7.2 at a flow rate of 0.5 mL/min (isocratic method for 30 min). Void and bed volumes were calibrated with λ-DNA (λ-DNA Molecular Weight Marker III 0.12–21.2 Kbp, Roche) and sodium azide (Merck), respectively. Column void volume (T₀): 10.58 min; total volume (T_tot): 23.03 min. Distribution coefficient, Kd = (T_retention—T₀)/(T_tot—T₀). Polysaccharide peaks were detected by differential refractive index (dRI) or at 214 nm, when run with a dextran standard curve (270–12 kDa range) to calculate the apparent average molecular weight. Exopolysaccharide was fractionated and collected as follow: HMW-PS, from 11.5 min to 13 min; MMW-PS, from 14 min to 16 min; LMW-PS, from 17 min to 18 min.

Conjugate, free protein and free GACox samples were eluted on a TSK gel G3000 PW_XL (30 cm × 7.8 mm) column (particle size 7 µm) with TSK gel PW_XL guard column (4.0 cm × 6.0 mm; particle size 12 µm) (TosohBioscience, King of Prussia, PA, USA). The mobile phase was 0.1 M NaCl, 0.1 M NaH₂PO₄, 5% CH₃CN, pH 7.2 at the flow rate of 0.5 mL/min (isocratic method for 35 min). Sample volume of injection was 80 µL. Void and bed volume calibration was performed with λ-DNA (λ-DNA Molecular Weight Marker III 0.12–21.2 Kbp, Roche, Risch-Rotkreuz, Switzerland) and sodium azide (NaN₃, Merck, Darmstadt, Germany), respectively. GACox peaks were detected by refractive index (RI). Protein and conjugate peaks were also detected using tryptophan fluorescence (emission spectrum at 336 nm, with excitation wavelength at 280 nm). For the Kd determination the following equation was used: $\mathrm{Kd}=\frac{\left(\mathrm{Te}-\mathrm{T}0\right)}{\left(\mathrm{Tt}-\mathrm{T}0\right)}$
where: Te = elution time of the analyte, T0 = elution time of the bigger fragment of λ-DNA and Tt = elution time of NaN₃.