To measure a hydrodynamic radius of eEF1Bα we used the Dynamic Light Scattering technique (DLS) and the Adaptive Correlation approach described in (22 (link)). DLS experiments were performed using a Zetasizer Nano ZS (Malvern Panalytical Ltd, UK) at 25°C with a scattering angle of 173° in air. All samples (1 ml) were measured in a 1 cm glass cuvette. Briefly, three measurements for each eEF1Bα concentration were done. Each measurement included 30 sub-measurements with duration time of 1, 2 and 3 s. The steady-state sub-measurements were analyzed using cumulants analysis. The correlation functions of the steady-state sub-measurements were averaged to report the hydrodynamic radius (Zave) and polydispersity index (PdI) values. Zave values were obtained for four different eEF1Bα concentrations. RH0 – a hydrodynamic radius of eEF1Bα at the infinite dilution was calculated from the plot of Zave versus eEF1Bα concentration by extrapolation to zero concentration.
Prior to the DLS measurements, eEF1Bα was subjected to gel filtration on a Sephacryl S200 column (GE Healthcare) equilibrated in buffer, containing 25 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5% glycerol (v/v) and 1 mM dithiothreitol. Fractions with the highest eEF1Bα concentration were centrifuged at 16 000 g for 2.5 hours at 10°C. Buffer was prepared using ultrapure deionized water, filtered (pore size 0.2 μm) and degassed.
Prior to the DLS measurements, eEF1Bα was subjected to gel filtration on a Sephacryl S200 column (GE Healthcare) equilibrated in buffer, containing 25 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5% glycerol (v/v) and 1 mM dithiothreitol. Fractions with the highest eEF1Bα concentration were centrifuged at 16 000 g for 2.5 hours at 10°C. Buffer was prepared using ultrapure deionized water, filtered (pore size 0.2 μm) and degassed.
