Multi wavelength absorbance detector

SEC-MALS measurements for CckA^Rec were performed at a sample loading concentration of 13.8 mg/ml; 6.9 mg/ml and 3.5 mg/ml at 25 C in sample buffer using a GE Healthcare Superdex 200 10/300 Increase column on an Agilent 1260 HPLC. Elution was monitored using an Agilent multi-wavelength absorbance detector (data collected at 280 and 254 nm), a Wyatt Heleos II 8+ multi-angle light scattering detector and a Wyatt Optilab rEX differential refractive index detector. The column was equilibrated overnight in the running buffer to obtain stable baseline signals from the detectors before data collection. Inter-detector delay volumes, band broadening corrections, and light-scattering detector normalization were calibrated using an injection of 2 mg/ml BSA solution (ThermoPierce) and standard protocols in ASTRA 6. Weight-averaged molar mass (Mw), elution concentration, and mass distributions of the samples were calculated using the ASTRA 6 software (Wyatt Technology).

SEC-MALS experiments were performed using a Superdex Increase 200 10/300 GL column (GE Healthcare) on an Agilent 1260 HPLC Infinity II in PBS buffer at RT (~297 K). Protein elution was monitored by three detectors in series namely, an Agilent multi-wavelength absorbance detector (absorbance at 280 nm and 254 nm), a Wyatt miniDAWN TREOS multiangle light scattering (MALS) detector, and a Wyatt Optilab rEX differential refractive index (dRI) detector. The column was pre-equilibrated overnight in the running buffer to obtain stable baseline signals from the detectors before data collection. Molar mass, elution concentration, and mass distributions of the samples were calculated using the ASTRA 7.1.3 software (Wyatt Technology). A BSA solution (2–4 mg/ml), purchased from Sigma-Aldrich and directly used without further purification, was used to calibrate inter-detector delay volumes, band broadening corrections, and light-scattering detector normalization using standard protocols within ASTRA 7.1.3.

SEC–MALS measurements were performed at 25 °C in 25 mM HEPES (pH 7.5), 500 mM NaCl and 1 mM DTT as the column buffer using a GE Healthcare Superdex 200 10/300 Increase column on an Agilent 1260 HPLC at a flow rate of 0.5 ml min^–1. Loading concentrations were 200 µM for the SOA(1–112) and SOA(1–229) fragments and 11 µM for the SOA(1–331) fragment. Elution was monitored using an Agilent multi-wavelength absorbance detector (data collected at 280 and 260 nm), a Wyatt Heleos II 8+ multi-angle light scattering detector and a Wyatt Optilab differential refractive index detector. The column was equilibrated overnight in the running buffer to obtain stable baseline signals from the detectors before data collection. Inter-detector delay volumes, band-broadening corrections and light-scattering detector normalization were calibrated using an injection of 2 mg ml^–1 BSA solution (Thermo Pierce) and standard protocols in ASTRA 8. Weight-averaged molar mass (M_w), elution concentration and mass distributions of the samples were calculated using ASTRA 8 software (Wyatt Technology).