Superdex 200 10 300 analytical column

The molecular weight and homogeneity of the sample was checked using a SEC column coupled with multi-angle laser light scattering (MALLS) Dawn DSP detector (Wyatt Technology, Santa Barbara, CA, USA). A GE Healthcare Superdex 200 10/300 analytical column was pre-equilibrated with the sample buffer, 25 mM HEPES pH 7.5, 150 mM NaCl, 5% Glycerol, 2 mM MgCl₂, 2 mM TCEP. The system was operated at 20°C, with a flow rate of 0.75 mL.min^-1.

The molecular weight and homogeneity of the sample was checked using a SEC column coupled with MALLS Dawn DSP detector (Wyatt Technology, Santa Barbara, CA, USA) (Figure 1B). To prevent bacterial growth, 0.01% sodium azide was added to the resuspension buffer, which was then filtered through 0.25 µm filter membranes (Millipore) before equilibrating the Superdex 200 10/300 analytical column (GE healthcare life sciences). The system was operated at 20°C, with a flow rate of 0.75 ml/min.

OdinAK elution peak was further analyzed by SEC–multiangle light scattering (MALS) using an ÄKTApure system (GE Healthcare) coupled to a miniDAWN TREOS II detector and an OptiLab T-rEX online refractive index detector (Wyatt Technology). The absolute molar mass was calculated by analyzing the scattering data using ASTRA analysis software package, version 7.2.2.10 (Wyatt Technology). Bovine serum albumin (BSA) was used for calibration, and proteins were separated on a Superdex 200 10/300 analytical column (GE Healthcare) with a flow rate of 0.4 ml/min. Sample (200 μl) at 2 mg/ml was injected and eluted in 30 mM Mops (pH 7) and 150 mM NaCl buffer. The refractive index increment of Odin was set at 0.185 ml/g, and the extinction coefficient for ultraviolet detection at 280 nm was calculated from the primary sequence. The calculated molecular weight is the average from three runs, with the error being the SD.