Pd minitrap sephadex g 25 columns

For coupling of the sdAb to the liposomal surface, the buffer was exchanged to coupling buffer (PBS, pH 7.0) with PD MiniTrap™ Sephadex G-25 columns (GE Healthcare, Opfikon, Switzerland). A sdAb to lipid ratio of 0.4 nmol/μmol was chosen for the reaction, resulting in approximately 30 sdAb per liposome, as described previously [48 (link)]. The reaction was incubated overnight at 4 °C and non-coupled sdAb were removed by two steps of washing and filtration via spin filter centrifugation (Amicon Ultra 0.5, 100 kDa, Merck Millipore, Schaffhausen, Switzerland). The mean diameter and polydispersity index (PDI) of liposomes were measured by dynamic light scattering (Litesizer 500, Buchs, Switzerland). To estimate the amount of sdAb coupled to the liposomes, gel electrophoresis was performed with labelled liposomes and defined amounts of the corresponding sdAb under denaturing and reducing conditions. Sample separation, Western blotting and imaging were performed as described above with anti-6xHis-tag antibody (ab 18184, Abcam, Cambridge, UK).

NWC_sup (0.5 mL) were desalted on PD MiniTrap Sephadex G-25 columns (GE Healthcare UK Limited, Buckinghamshire, UK), following the manufacturer instructions.
The electrophoretic separation of WPs was performed under both reducing and non-reducing conditions, using the Agilent Protein 80 Kit on an Agilent 2100 Bioanalyzer equipped with 2100 Expert Software (Agilent Technologies, Inc., Santa Clara, CA, USA), according to the instructions of the provider.