Affinity Purification of Recombinant IRE1α Protein

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)].