Vivaspin ultrafiltration tubes

Encapsulation efficiency of nanocapsule formulations was measured as described before (24 (link)). Shortly, encapsulation efficiency was calculated by measuring the total fluorescence intensity of PEG-Rhodamine (λex = 553 nm and λem = 576 nm) in the aqueous suspension. Afterwards, the samples were centrifuged and the remaining, unencapsulated PEG-Rhodamine in the recovered suspension as well as the PEG-Rhodamine in the redispersed NCs was measured.
Release of payload from NCs was measured as previously described (24 (link)). Shortly, the release of the PEG-Rhodamine was analyzed by centrifugation of NCs at 1,770 g for 30 min using Vivaspin® Ultrafiltration tubes (500 μl 1,000 K; Sartorius AG, Germany) and measuring the fluorescence of the filtrate (λex = 553 nm and λem = 576 nm).

Purified LPMO solutions were concentrated to 0.5 ml volume using Vivaspin ultrafiltration tubes (10 kDa MWCO, Sartorius, Göttingen, Germany) and incubated with two-fold molar surplus of Cu(II)SO₄ at room temperature for 30 min. The excess (unbound) copper was removed by desalting with a PD MidiTrap G-25 gravity flow column (GE Healthcare, Chicago, USA), equilibrated with 50 mM sodium phosphate buffer, pH 6.0. To ensure effective free copper removal, the ScLPMO10C and ScLPMO10C_TR samples were then subjected to multiple consecutive rounds of dilution and concertation using 50 mM sodium phosphate buffer, pH 6.0 and Vivaspin ultrafiltration tubes. The resulting total dilution factors for solutions of copper-saturated LPMOs amounted to at least 160 000.