SEC and AF4 methods

UV detection was at 280 nm unless stated otherwise, and the temperature of the autosampler was set at 5 °C to prevent protein degradation or aggregation during experiments. For SEC experiments, the injection volume was 10 μL and the flow rate was 0.3 mL/min. For the AF4 experiments, the sample was focused for 3 min with a focus flow of 1.5 mL/min and the focusing point was set at 16% of the channel length, which corresponded to a focusing position of 2.7 cm from the channel inlet. The injection inlet was permanently closed with a flat bottom plug and during focusing the sample was injected from the inlet. The elution of the sample was performed in the constant cross-flow mode. Pretreatment with saturation of 50 μg BSA was performed for every new membrane once before experiments to enhance recovery. The injected mass for the recovery experiments and the measurements of the diffusion coefficients was 10 μg (10 μL injection volume). For the overloading experiments, the injected mass was 10 μg and 50 μg unless stated otherwise.