Histrap ff nickel columns

Cell pellets were defrosted and resuspended in Buffer A (20 mm Imidazole pH 7.0, 400 mM NaCl and 0.05% Tween 20) with lysozyme. Cells were lysed on ice by sonication and lysate was clarified by centrifugation at 48 400 g for 1 h (4°C). Supernatant was loaded onto buffer A equilibrated HisTrap FF nickel columns (GE Healthcare). Columns were washed with 12 CVs of buffer A before two 20 ml elution steps at 8% buffer B (400 mm Imidazole pH 7.0, 400 mM NaCl and 0.05% Tween 20) in buffer A and 100% buffer B. The purity of samples was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Eluted protein was dialysed overnight at 4°C against 20 mM HEPES pH 7.0 and 200 mM NaCl. Protein was concentrated to 40 mg ml⁻¹ using Vivaspin spin-concentrators (Sartorius) and snap-frozen.

Cell pellets were thawed, resuspended, and lysed in ‘cytobuster’ (EMD Millipore) containing protease (EDTA-free) and phosphatase inhibitor cocktails (Pierce) and 30 mM imidazole, pH 8.0. The resulting cell lysates were clarified and affinity purified on HisTrapFF nickel columns (GE Healthcare) using an AKTA FPLC (GE Healthcare, Marlborough, MA, USA). Bound protein was eluted in a linear gradient of elution buffer (50 mM sodium phosphate buffer, pH 7.2, 500 mM NaCl, 500 mM imidazole). Peak fractions were run on SDS-PAGE gels and analyzed by Coomassie staining. IRE1α containing fractions were pooled and concentrated in Amicon Ultra 30K cut off concentrators (EMD Millipore) and buffer was exchanged into 25 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM DTT, 5% glycerol. Protein concentration was determined by Bradford assay. Protein was aliquoted and stored at −80 °C, with additional glycerol (25% final) added for longer term storage. This expression and purification approach has been successfully used to study IRE1α kinase activity in vitro [58 (link),60 (link),72 (link)].