Astra 7

An Agilent 1260 II HPLC system (Agilent Technologies), which consists of a variable wavelength detector, a quaternary pump, an online vacuum degasser, and an autosampler, was used in tandem with a multiangle light scattering (MALS) detector (DAWN‐HELEOS; Wyatt Technology), and a differential refractive index detector (Optilab^® T‐Rex; Wyatt Technology). The mobile phase was 50 mM Na‐phosphate/100 mM NaCl, pH 7.4 (PBS). Prior to injection, the protein samples were passed through a membrane filter (pore size 0.1 μm; Supelco Analytical). The sample volume injected into the channel was 100 μl, and the flow rate was set at 0.5 ml/min. Data acquisition and evaluation were accomplished using Astra 7 software (Wyatt Technology Corporation).
The stoichiometry of binding between the AGL55 and K2_hPg in the two‐component complex was determined using the Protein Conjugate Analysis Method in Astra 7 software (Wyatt Technology). This method, based on the unique specific refractive index increments (dη/dc) and extinction coefficients of each protein enables accurate determination of the components of the complex along molecular mass distributions.

SEC-MALS experiments were performed using a Superdex Increase 200 10/300 GL column (GE Healthcare) on an Agilent 1260 HPLC Infinity II in PBS buffer at RT (~297 K). Protein elution was monitored by three detectors in series namely, an Agilent multi-wavelength absorbance detector (absorbance at 280 nm and 254 nm), a Wyatt miniDAWN TREOS multiangle light scattering (MALS) detector, and a Wyatt Optilab rEX differential refractive index (dRI) detector. The column was pre-equilibrated overnight in the running buffer to obtain stable baseline signals from the detectors before data collection. Molar mass, elution concentration, and mass distributions of the samples were calculated using the ASTRA 7.1.3 software (Wyatt Technology). A BSA solution (2–4 mg/ml), purchased from Sigma-Aldrich and directly used without further purification, was used to calibrate inter-detector delay volumes, band broadening corrections, and light-scattering detector normalization using standard protocols within ASTRA 7.1.3.

High-performance size-exclusion chromatography system (HPSEC) was applied for the molecular parameter analysis of WJP-F80 according to our previous methods [24 (link)]. Sample was dissolved in NaNO₃ solution (0.1 M) and filtered for injection. An OHpak SB-803 HQ column and an OHpak SB-805 HQ column (8 mm × 300 mm, Shodex, Tokyo, Japan) in series were used for separation, and 0.1 M NaNO₃ solution containing 0.02 wt% NaN₃ was applied as eluent with a flow rate of 0.6 mL/min. Three detectors, a multi-angle laser light scattering detector (DAWN HELEOS-II, MALLS), a differential pressure viscometer (ViscoStar III, DP), and a refractive index detector (Optilab T-Rex, RI) (Wyatt Technology, Santa Barbara, CA, USA) in series were equipped with the HPLC system. For the data collection and analysis, ASTRA 7.1.3 software (Wyatt Technology, Santa Barbara, CA, USA) was applied, and a DN/DC value of polysaccharide sample was adopted as 0.145 for the calculation of molecular parameters [25 (link)].