2.1. Purification of BSA on an ion-exchange column  

For the preparative offline IEC experiment, 100 µl of a 77 mg ml⁻¹ BSA (lyophilized powder, essentially globulin-free; Sigma) solution in buffer A [20 mM Tris pH 7, 25 mM NaCl, 5% glycerol, 1 mM dithiothreitol (DTT)] was prepared and injected onto a Uno Q-1R (Bio-Rad) column on a Biologic system (Bio-Rad) equilibrated with buffer A. The protein concentration was determined by measuring the absorption at 280 nm with a NanoDrop (NanoDrop 1000, Thermo Fisher) using a mass extinction coefficient of 6.7 for a 1% (10 mg ml⁻¹) BSA solution. The high concentration enables easy injection onto the column using injection loops. A salt gradient was made by mixing buffer A with buffer B (20 mM Tris–HCl pH 7, 1 M NaCl, 5% glycerol, 1 mM DTT). The flow rate for the offline experiment was 2 ml min⁻¹.

For IEC–SAXS experiments, only 50 µl was injected using the autosampler of the HPLC system (SIL-20ACXR) and the flow rate was 1 ml min⁻¹.

Peak fractions from the preparative run (20 µl per 1500 µl fraction size) were analysed on a 12% SDS–PAGE stained with InstantBlue (Expedeon). PageRuler Plus Prestained Protein Ladder (Thermo Fisher) was used as a molecular-weight marker.