Minidawn treos 2 mals

The protein size was measured by injecting 200 µl 1 mg ml⁻¹ RsbS into a Superdex 200 analytical column with an ÄKTApurifier FPLC (GE Healthcare) and the elution products were analyzed using inline miniDAWN TREOS II MALS and Optilab rEX differential refractive-index detectors (Wyatt Technology, Santa Barbara, California, USA). The data were analyzed using the ASTRA 6 software package (Wyatt Technology).

Size-exclusion chromatography combined with multi-angle light scattering was performed to determine the homogeneity and the native molecular mass of all proteins under study. Analyses were performed on an LC20 Prominence HPLC equipped with a refractive index detector RID-10A and the photodiode array detector SPD-M20A (all from Shimadzu, Japan). In-line MALS was analyzed either with a miniDAWN TREOS II MALS (for analysis of Spike) or a Heleos Dawn8+ plus QELS apparatus (Wyatt Technology, United States). Prior to analysis, all proteins were centrifuged (16,000 g, 10 min, 20 °C) and filtered (0.1 µm Ultrafree-MC filter, Merck Millipore). Proper performance of the MALS detectors was validated with bovine serum albumin. Purified Spike was analyzed by injection of a total of 50 µg onto a Superose 6 Increase 10/300 GL column (Cytiva) at a flow rate of 0.25 mL min^–1. The mobile-phase buffer used was PBS supplemented with 10% glycerol (pH 7.4). All other proteins were analyzed by using a Superdex 200 10/300 GL column (Cytiva) equilibrated with PBS plus 200 mM NaCl (pH 7.4). A total of 25 µg of each protein was injected and experiments were performed at a flow rate of 0.75 mL min^–1. Data were analyzed using the ASTRA six software (Wyatt Technology).