Lc20 ad isocratic pump system

Experiments were conducted on a system comprising a Wyatt HELEOS-II multiangle light scattering detector and a Wyatt rEX refractive index detector linked to a Shimadzu liquid chromatography (LC) system (SPD-20A UV detector, LC20-AD isocratic pump system, DGU-20A3 degasser, and SIL-20A autosampler). Experiments were conducted at room temperature (20 ± 2 °C). Solvents were filtered through a 0.2 µm filter prior to use, and a 0.1 µm filter was present in the flow path. The column was equilibrated with >2 CV of buffer (50 mM NaPi, 300 mM NaCl pH 7.4) before use and buffer was infused at the working flow rate until baselines for UV, light scattering, and refractive index detectors were all stable. The sample injection volume was 100 µL of protein at 6 mg mL⁻¹ in 50 mM NaPi buffer, 300 mM NaCl pH 7.4. Shimadzu LC Solutions software was used to control the LC and Astra V software for the HELEOS-II and rEX detectors. The Astra data collection was 1 min shorter than the LC solutions run to maintain synchronization. Blank buffer injections were used as appropriate to check for carryover between sample runs. Data were analyzed using the Astra V software. Molecular weights were estimated using the Zimm fit method with degree 1. A value of 0.158 was used for protein refractive index increment (dn/dc).