Astra six software

The absolute molar masses of protein samples and complexes were determined by size-exclusion chromatography multi-angle light scattering (SEC-MALS). Protein samples at >1 mg/ml (unless otherwise states) were loaded onto a Superdex 200 Increase 10/300 GL size exclusion chromatography column (GE Healthcare) in 20 mM Tris pH 8.0, 150 mM KCl, 2 mM DTT, at 0.5 ml/min using an ÄKTA Pure (GE Healthcare). The column outlet was fed into a DAWN HELEOS II MALS detector (Wyatt Technology), followed by an Optilab T-rEX differential refractometer (Wyatt Technology). Light scattering and differential refractive index data were collected and analysed using ASTRA six software (Wyatt Technology). Molecular weights and estimated errors were calculated across eluted peaks by extrapolation from Zimm plots using a dn/dc value of 0.1850 ml/g. SEC-MALS data are presented as differential refractive index (dRI) profiles with fitted molecular weights (M_W) plotted across elution peaks.

SEC-MALS was used to determine the absolute molecular masses of full-length CtIP WT and mutants. Approximately 50 µg samples of CtIP were loaded at 0.5 ml/min onto a Superose 6 10/300 size-exclusion chromatography column (GE Healthcare) in 20 mM Tris, pH 8.0, 200 mM NaCl, 1 mM TCEP using an Agilent HPLC. The eluate from the column was coupled to a DAWN HELEOS II MALS detector (Wyatt Technology) and an Optilab T-rEX differential refractometer (Wyatt Technology). ASTRA six software (Wyatt Technology) was used to collect and analyse light scattering and differential refractive index data according to the manufacturer’s instructions. Molecular masses and estimated errors were calculated across individual eluted peaks.

Size-exclusion chromatography combined with multi-angle light scattering was performed to determine the homogeneity and the native molecular mass of all proteins under study. Analyses were performed on an LC20 Prominence HPLC equipped with a refractive index detector RID-10A and the photodiode array detector SPD-M20A (all from Shimadzu, Japan). In-line MALS was analyzed either with a miniDAWN TREOS II MALS (for analysis of Spike) or a Heleos Dawn8+ plus QELS apparatus (Wyatt Technology, United States). Prior to analysis, all proteins were centrifuged (16,000 g, 10 min, 20 °C) and filtered (0.1 µm Ultrafree-MC filter, Merck Millipore). Proper performance of the MALS detectors was validated with bovine serum albumin. Purified Spike was analyzed by injection of a total of 50 µg onto a Superose 6 Increase 10/300 GL column (Cytiva) at a flow rate of 0.25 mL min^–1. The mobile-phase buffer used was PBS supplemented with 10% glycerol (pH 7.4). All other proteins were analyzed by using a Superdex 200 10/300 GL column (Cytiva) equilibrated with PBS plus 200 mM NaCl (pH 7.4). A total of 25 µg of each protein was injected and experiments were performed at a flow rate of 0.75 mL min^–1. Data were analyzed using the ASTRA six software (Wyatt Technology).