Superdex 200 increase 3.2 300

The oligomerization states of DfrB-H5 WT and W236F were analysed using analytical SEC with an ÄKTA fast protein liquid chromatography system. The 2.4 ml size exclusion column (Superdex 200 Increase 3.2/300, Cytiva) was calibrated with the Cytiva Gel Filtration Calibration Kit. Different protein concentrations (10 µl injections) were applied onto the column equilibrated with 50 mM potassium phosphate, pH 8, at a flow rate of 0.075 ml min⁻¹.

Analyses were performed of 20 μM (HACE1), 80 μM (RAC1) or mixtures thereof (molar ratio, 1:4) in 50 mM HEPES (pH 8.0), 150 mM NaCl and 1 mM DTT (Superdex 200 Increase 3.2/300; Cytiva), using an ÄKTA Micro (Cytiva) at 4 °C.

SEC-MALS experiments were performed using a Superdex 200 Increase 3.2/300 size exclusion column on an AKTA FPLC system (Cytiva) coupled to inline static light scattering (Dawn Heleos II, Wyatt Technology), differential refractive index (Optilab rEX, Wyatt Technology), and UV detection. Purified cPcdh proteins were diluted to 18 μM in running buffer (150 mM NaCl, 10 mM Tris–Cl, pH 8, 3 mM CaCl₂, 200 mM imidazole, pH 8) and 50 or 100 μl samples were run at a flow rate of 0.5 ml/min at room temperature. Mixtures of cPcdh fragments were prepared in the same buffer at final concentrations of 18 μM for each protein and run under the same conditions. Data were analyzed using ASTRA software (Wyatt Technologies).
During SEC-MALS experiments, a dimer/monomer equilibrium is established as proteins move through the size exclusion chromatography column, which is influenced by the K_D of the interaction. The concentrations used in the current experiments (18 μM for each cPcdh fragment), although above the K_D of 3 μM for the γC3/γA4 cis interaction, are not sufficiently high for all the cis fragments to be bound into heterodimers, leaving a significant population of molecules as monomers, resulting in apparent molecular weights of ~76 kDa for the dimeric species compared to the predicted molecular weight for a dimer of ~108 kDa.