Streptactin superflow cartridge

Purification of the protein complexes were performed at 4 °C. Cells were resuspended in lysis buffer [50 mM Tris/HCl (pH 8.3), 250 mM NaCl, 1 mM EDTA, 1 mM DTT, Protease inhibitor cocktail tablets (PIC) (Complete EDTA-free; Roche Diagnostics GmbH), 0.1 mM PMSF, 5 units/ml benzonase (Novagen)], subsequently sonicated and centrifuged for 1 h at 48,000 g. The soluble fraction was slowly (1 ml/min flow rate) applied to a 5 ml StrepTactin Superflow Cartridge (Qiagen) and washed with wash buffer (lysis buffer without PIC, PMSF and benzonase) until stable UV absorption could be observed. Peak fractions were incubated with TEV protease at 4 °C overnight and wash buffer without NaCl (buffer A) was used for a two-fold dilution before loading onto a ResourceQ anion-exchange column (GE Healthcare) the next day. After a washing step the complexes were eluted using a gradient with buffer B [20 mM Tris/HCl (pH 8.0), 1 M NaCl, 1 mM DTT]. A final size exclusion step on a Superose 6 Increase 10/300 GL column with 20 mM Hepes-NaOH (pH 7.8), 200 mM NaCl and 1 mM DTT was performed.