Optilab refractometer

Analytical SEC measurements were performed coupled to a miniDAWN Multi Angle Light Scattering (MALS) detector and an Optilab refractometer (Wyatt Technology). Samples previously centrifuged and filtered were run in a Superdex 75 Increase 10/300 GL column connected to an Äkta Pure System (GE Healthcare Life Sciences) equilibrated with buffer 10 mM sodium phosphate, 50 mM sodium chloride, 0.02% sodium azide, pH 7.8. Experiments were conducted at room temperature with a protein concentration of 1 and 0.8 mL min⁻¹ flow rate. For the samples containing ribose, 0.5 mM of ribose was premixed with protein at 1 mg mL⁻¹. Reproducibility during all SEC‐MALS measurements was tested by running a BSA standard at 2 mg mL⁻¹ at the beginning and end of all experiments, which resulted in identical data. Determination of weight averaged molar mass was performed by using the Zimm‐Equation with the differential refractive index signal as source for the concentration calculations (refractive index increment dn/dc set to 0.185). Data collection and analysis were done using the ASTRA v.7.3.2 software (Wyatt Technology).

A 100-µL sample of BxpB was injected onto a WTC-MP015N5 column, 4.6 by 300 mm I.D; particle size, 5 mm; pore size, 150 Å (Wyatt Technology, Santa Barbara, CA) on a Shimadzu Prominence HPLC System (Shimadzu Corp., Kyoto, Japan) with an isocratic run at 0.3 mL/min for 17 min, and a solution of 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 2 mM CaCl2, and 2 mM TCEP was used as the mobile phase. UV absorbance was set at 280 nm using Shimadzu HPLC Explorer software (Shimadzu Corp., Kyoto, Japan). A DAWN 8 MALS detector (Wyatt Technology, Santa Barbara, CA), set at 659 nm, and an Optilab refractometer (Wyatt Technology, Santa Barbara, CA) were used in tandem for detection. Bovine serum albumin (Wyatt Technology, Santa Barbara, CA) was used to normalize the static light scattering detector. The delay volume, band broadening parameters, and the light scattering and differential refractive index measurements were analyzed using Astra 8 software (Wyatt Technology, Santa Barbara, CA).

Size-exclusion chromatography–multi-angle light scattering (SEC-MALS) measurements were performed with a miniDAWN detector and an Optilab refractometer (Wyatt Technology) coupled to an analytical size-exclusion chromatography column (Superdex 75 Increase 10/300 GL). Centrifuged samples were run on the column connected to an ÄKTApure FPLC system (GE Healthcare Life Sciences) and equilibrated with 10 mM sodium phosphate pH 7.8, 50 mM sodium chloride, 0.02% sodium azide at room temperature. Measurements were run at a constant flow rate of 0.8 ml min⁻¹ at protein concentrations of 0.5, 1.0 and 5 mg ml⁻¹. The system setup was normalized and checked by measurement of a commercially available standardized BSA sample (2 mg ml⁻¹; Pierce, catalogue No. 23209) before and after each series of measurements. Weight-averaged molar-mass determination was performed using the Zimm equation with the differential refractive-index signal as a source for the concentration calculations (the refractive-index increment dn/dc was set to 0.185). Analysis of the experiments was performed using the ASTRA version 7.3.2 software suite (Wyatt Technology).