Optilab system

Size exclusion chromatography (SEC) coupled with multiangle light scattering (MALS) was performed by injecting 150 μg of activated or unactivated probe 3 or 4 onto a WTC-010S5 column (Wyatt Technology) previously equilibrated in PBS buffer at a flow rate of 0.5 mL/min. The elution profile was monitored by light scattering at 658 nm (HELEOS system, Wyatt Technology) and differential refractometry (Optilab system, Wyatt Technology). UV Absorbance was not used due to saturation of the detector at the concentrations above the detection limit of light scattering. Data analyses were carried out using ASTRA 6.0 software (Wyatt Technology). The refractive index increment (dn/dc) value of 0.163 mLμg⁻¹ was assumed based on literature,^{84 (link)} as experimental determination was hindered by limited solubility of samples. Calculated molecular weights were further confirmed by MALDI-TOF mass spectrometry. SEC traces of polymerization reactions of different concentrations at different time points were plotted using Origin Lab 8.5 as 3D stacking and 2D intensity graphs. Different species (monomer, polymer species around 25 kDa alone or with other oligomers) were quantified using integration of area under curve (AUC) of the differential refractometry data and plotted against time using Origin Lab 8.5.

SEC–MALS was performed by injecting 25 μg of purified recombinant ATG14 on a WTC-030S5 column (Wyatt Technology) that was previously equilibrated in 10 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, pH 8.0 at a flow rate of 0.5 ml min⁻¹. The eluted sample was monitored by ultraviolet absorption at 280 nm (Jasco UV-975 ultraviolet–visible system), light scattering at 658 nm (HELEOS system, Wyatt Technology) and differential refractometry (Optilab system, Wyatt Technology). The data analyses used ASTRA 6.0 software (Wyatt Technology). The protein absolute molecular mass was calculated in ASTRA 6.0 (Wyatt Technology) assuming a dn/dc value of 0.185 ml g⁻¹ and a theoretical ultraviolet extinction coefficient value of 1.0 ml mg⁻¹ cm⁻¹.