Optilab rex refractive index monitor

SEC-MALLS analysis of DnaA^DI and SirA–DnaA^DI was carried out at a range of protein concentrations: DnaA^DI was analysed at 1.0 mg ml⁻¹, 2.5 mg ml⁻¹ and 5.0 mg ml⁻¹ and SirA–DnaA^DI was analysed at 0.5 mg ml⁻¹, 1.0 mg ml⁻¹ and 2.5 mg ml⁻¹. For each run, 100 μl of sample was loaded onto a Superdex 75 HR 10/30 size-exclusion column equilibrated with 50 mM Tris pH 8.5, 200 mM KCl at a flow rate of 0.5 ml min⁻¹. The eluate was analysed successively by a SPD20A UV/Vis detector, a Wyatt Dawn HELEOS-II 18-angle light scattering detector and a Wyatt Optilab rEX refractive index monitor as described previously (Colledge et al., 2011 (link)). Data were analysed with Astra software (Wyatt).

For determination of the state of these proteins, human RBPMS2-Nter and RBPMS2-Nter-L49E were analyzed using an HPLC-MALS apparatus. We loaded 0.05-ml samples of protein at ∼2–4 mg/ml onto a Superdex 75 10/300 gel-filtration column equilibrated at 0.5 ml/min with a mobile phase consisting of 20-mM Tris–HCl at pH 8 and 300-mM sodium chloride. The eluate was passed successively through a UV 900 GE monitor, a Wyatt Optilab rEX refractive index monitor and a Wyatt miniDawn TREOS 3-angle light-scattering detector with the system driven by an AKTA purifier HPLC system comprising a P900 pump. The data were processed, and molecular masses were calculated using the Astra 6.1 software (Wyatt).

Samples were prepared at 2 mg/mL in 100 μL in 50 mM Tris-HCl, 150 mM NaCl pH 8.0 and injected into a Superdex 200 HR 10/30 column (GE Healthcare) at 25 °C at a flow-rate of 0.5 mL/min connected to a Shimadzu HPLC system comprising LC-20AD pump, SIL-20AC autosampler and SPD20A UV/Vis detector with 50 mM Tris-HCl, 150 mM NaCl pH 8.0 as running buffer. Light scattering was detected by a Wyatt Dawn HELEOS-II 8-angle light scattering detector and Wyatt Optilab rEX refractive index monitor. The resulting light scattering, refractive index and UV traces were processed in ASTRA 6 (Wyatt Technologies).

100-μl samples of TolR constructs at various protein concentrations were loaded onto a Superdex75 10/300 (GE Healthcare) gel filtration column connected to a Shimadzu HPLC and equilibrated in 50 mm Tris, pH 7.5, 150 mm NaCl at a flow rate of 0.5 ml/min. Elution was monitored with a DAWN HELEOS-II 8-angle light scattering detector (Wyatt Technology), a SPD-20A UV/VIS detector (Shimadzu), and an OPTILab rEX refractive index monitor (Wyatt Technology). Data were analyzed with the program Astra 6 (Wyatt Technology), and molar masses of tested constructs were calculated.

SEC was performed on Superdex 200 10/300 column (GE Healthcare) equilibrated in 20 mM Tris‐HCl pH7.5, 150 mM NaCl. 100 μl of protein was injected at increasing concentrations and eluted at 0.4 ml min⁻¹. The column was followed in line by a Dawn Heleos‐II light scattering detector (Wyatt Technologies) and an Optilab‐Rex refractive index monitor (Wyatt Technologies). Molecular mass calculations were performed using ASTRA 6.1.1.17 (Wyatt Technologies) assuming a dn/dc value of 0.186 ml g⁻¹.