10 kda cut off centrifugal concentrator

Once bound to the initial Ni-NTA and washed with 10 cv A500, the column was washed with a further 5 cv A100. The sample was eluted directly on to a pre-equilibrated anion exchange column with 10 cv B100 before washing again in A100 to remove the high imidazole. The anion exchange column was run as above. Fractions were analysed for protein purity by SDS-PAGE, and appropriate fractions were pooled before the addition of 0.4 mg 6His-TEV protease to cleave the 6His-TEV site tag. The sample was rolled overnight at 4°C then passed down a second Ni-NTA column (ortho Ni-NTA) to remove the 6His-TEV protease and 6His-TEV site tag. The flow through was collected and concentrated in a 10 kDa cut-off centrifugal concentrator (Sartorius) to 2 ml. The 2 ml sample was injected into a 2 ml capillary loop on the Åkta™ Pure system before fractionation by SEC using the S-300 column, as above. Fractions were analysed for purity by SDS-PAGE, appropriate fractions were pooled and concentrated to >300 μM before diluting by one third volume with storage buffer for a final glycerol (cryoprotectant) concentration of 30%, and final protein concentration of >200 μM. Appropriate volume aliquots were made, and flash cooled in liquid nitrogen before storage at −80°C.