Endogenous clathrin coated vesicles were extracted from Sus scrofa brains and clathrin purified from them as triskelia using previously described methods 64 (link). The initial assembly for harvesting cages was performed by dialysis into polymerisation buffer pH 6.2 and subsequent ultracentrifugation with concentration by resuspension of the pellet into a small volume of polymerisation buffer. All subsequent uses of polymerisation buffer utilised a pH of 6.4. Clathrin concentration was assayed by A280 of triskelia to avoid effects from light scattering.
The GST β2-adaptin616-951 plasmid was a kind gift from Steve Royle, University of Warwick 65 (link). β2-adaptin616-951 was expressed as a GST fusion protein in an E. coli BL21 strain and purified using GSH resin (GE Healthcare), the GST tag was subsequently removed by cleavage using a commercially available GST fusion 3C protease (Prescission, GE Healthcare) overnight at 4 °C in Prescission buffer. Cleavage enzyme was removed by GSH resin and the cleaved protein collected from the flow through after which it was concentrated and exchanged into Tris buffer on vivaspin columns (Sartorius).
The GST β2-adaptin616-951 plasmid was a kind gift from Steve Royle, University of Warwick 65 (link). β2-adaptin616-951 was expressed as a GST fusion protein in an E. coli BL21 strain and purified using GSH resin (GE Healthcare), the GST tag was subsequently removed by cleavage using a commercially available GST fusion 3C protease (Prescission, GE Healthcare) overnight at 4 °C in Prescission buffer. Cleavage enzyme was removed by GSH resin and the cleaved protein collected from the flow through after which it was concentrated and exchanged into Tris buffer on vivaspin columns (Sartorius).
