Monomeric bovine serum albumin

Recombinant SARS-CoV-2 RBDs hu-RBD and Lt-RBD (20 μl of 1 mg/ml) were injected into a Protein KW-802.5 analytical size-exclusion column (Shodex) and separated with a flow rate of 1 ml min^–1 in PBS using a high-pressure liquid chromatography system (Shimadzu Prominence). For MW characterization, light scattering was measured with a miniDAWN MALS detector (Wyatt), connected to a differential refractive index detector (Shimadzu RID-20A) for quantitation of the total mass, and to a UV detector (Shimadzu SPD-20A), for evaluation of the sole protein content. Chromatograms were collected and analyzed using the glycoconjugate analysis algorithm available in the ASTRA7 software (Wyatt, using an estimated dn/dc value of 0.185 ml/g for proteins and 0.140 ml/g for glycans). Calibration of the instrument was verified by injection of 10 μl of monomeric bovine serum albumin (3 mg/L) (Sigma-Aldrich).

For MALS studies on the S. rosetta hub, purified protein at ∼3.2 mg/ml was injected into a Superdex 200 10/300 analytical SEC column equilibrated overnight in gel filtration buffer (25 mM Tris at pH 8.0, 150 mM KCl, 1 mm TCEP, and 10% glycerol). The chromatography system was coupled to an 18-angle light scattering detector (DAWN HELEOS-II) and a refractive index detector (Optilab T-rEX) (Wyatt Technology). Data were collected every second and the flow rate was set to 0.5 ml/min. Data analysis was carried out using the program ASTRA (Wyatt Technology). Monomeric bovine serum albumin (BSA; Sigma) was used for calibration of the light scattering detectors and data quality control. Measurement was carried out at 25°C.

Size exclusion chromatography/multi-angle light scattering analyses were performed on an HPLC system (Agilent) coupled to a mini DAWN TREOS multi-angle light scattering and RefractoMax 520 refractive index detectors (Wyatt Technology). 60 μl (15 nmol) of SuhB were passed over a Superdex 200 increase 10/300 column (GE Healthcare) in 20 mM HEPES, pH 7.5, 50 mM NaCl, 0.02% (w/v) NaN₃ at a flowrate of 0.6 ml/min. Data were analyzed with the ASTRA 6.1 software (Wyatt Technology) using monomeric bovine serum albumin (Sigma-Aldrich) as a reference.

SEC-MALS measurements (De et al., 2010 (link)) were carried out by injecting purified proteins (100 µM) onto a Phenomenex gel filtration column pre-equilibrated with MALS buffer (25 mM Tris–HCl [pH 7.4] and 100 mM NaCl). The chromatography system was coupled to an 18-angle, static light scattering detector and a refractive index detector (DAWN HELEOS-II and Optilab T-rEX, respectively; Wyatt Technology, Santa Barbara, CA). Data were collected at 25°C every second at a flow rate of 1 ml/min and analyzed with the software ASTRA, yielding the molecular weight and mass distribution (polydispersity) of the samples. For data quality control and normalization of the light scattering detectors, monomeric bovine serum albumin (Sigma-Aldrich, St. Louis, MO) was used.