Dawn helios 2

A 100 μl sample was loaded on to a Superdex S200 Increase 10/300 GL gel-filtration column (GE Healthcare Life Sciences) preequilibrated with 50 mM HEPES pH 8.0, 0.3 M NaCl using a Fast Protein Liquid Chromatography system (ÄKTA Purifier, GE Healthcare Life Sciences). Before loading, concentrated samples were centrifuged at 13,000 rpm, 20 min, 4 °C to remove any residual debris or aggregate. A flow-rate of 0.5 ml/min was applied for a minimum of 25 ml and 0.5 ml fractions collected (Äkta Frac-920, GE Healthcare Life Sciences). Size Exclusion Chromatography Multi-Angle Light Scattering was carried out in identical conditions to analytical SEC experiments (UFLC, Shimadzu; Dawn Helios-II, Wyatt; Optilab T-rEX, Wyatt), and data were analyzed using Astra 6. To test fraction activity, 25 μl of each fraction (concentration not normalized) was pipetted into a white 384-well plate and incubated at 37 °C for 10 min. Following incubation, 10 μM Leu-Mec substrate diluted in 100 mM Tris pH 8.0, 1.0 mM CoCl₂ was added, and activity monitored for 60 min. Resultant activity across the 60-min time frame was expressed as a fluorescence units/min and plotted against corresponding fraction elution volume using Prism GraphPad 8.

SEC-MALS experiments enabled the accurate mass estimation of the linked complexes. The Superdex 200 Increase 10/300 GL column was used for SEC, which was connected to an Agilent HPLC unit with an 18-angle light scattering detector (Wyatt Dawn HELIOS II) and a refractive index detector (Wyatt Optilab T-rEX). The experiments were performed at room temperature. The column was equilibrated with A50 (50 mM NaCl, 50 mM Tris pH 8.0) buffer at 0.4 ml/min. Bovine serum albumin at 2 mg/ml was used to calibrate the system. The purified linked complexes (approximately 5 mg/ml, 110 μl) were consequently loaded to estimate the molecular weight of the eluted peaks. The Zimm model implemented in ASTRA software (https://www.wyatt.com/products/software/astra.html) was used for the curve fitting and estimation of molecular weights. GraphPad Prism (https://www.graphpad.com) was used to average molar mass from fitted plots.

The AF4 was performed using the separation system Eclipse AF4 from Wyatt Technology. The PAE sample was concentrated as 0.3 wt% (3 g/L) aqueous solution with 0.05 M NaNO₃ and injected into the channel equipped with a 5 k Dalton RC-membrane (Millipore). The detector flow was 0.5 ml/min. During the measurement an exponentially decreasing cross flow was used. The cross flow decreased from 4 ml/min to 0.1 ml/min over a period of 35 min. The separation system was equipped with a light scattering system (DAWN Helios-II) and a refractive index measurement system (Optilab T-rEX) both from Wyatt Technologies.

The freshly thawed wild-type or C57D/C146D mutant SOD1 protein in buffer containing 20 mM Tris (pH 8.0) and 150 mM NaCl was used for the SEC-MALS analysis. Each sample was subjected to size-exclusion chromatography (SEC) on a Superdex 200 increase 10/300 GL column (GE Healthcare). The molecular sizes and oligomerization states of SOD1 were measured by MALS (DAWN HELIOS II; Wyatt Technology, USA).

To ascertain the oligomerisation state of dIG proteins, SEC-MALS was performed in a Dawn Helios II apparatus (Wyatt Technologies) coupled to a SEC Superdex 75 Increase 10/300 column. The column was equilibrated with PBS or buffer B at 25 °C and operated at a flow rate of 0.5 mL/min. A total volume of 100–165 μL of protein solution at 1.0–3.0 mg/mL was employed for each sample. Data processing and analysis proceeded with Astra 7 software (Wyatt Technologies), for which a typical dn/dc value for proteins (0.185 mL/g) was assumed.