Ni2 nta superflow cartridge

BamHI, HindIII and T4 DNA ligase were all purchased from Takara Biotechnology Co., Ltd. (Dalian, China). A Ni²⁺-NTA Superflow Cartridge was purchased from Qiagen (Valencia, CA, USA). PD-10 desalting columns were obtained from Amersham Pharmacia Biotech, Inc. (Piscataway, NJ, USA). Coomassie Brilliant Blue R-250 was obtained from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Anti-COX-1 (sc-166573) and anti-COX-2 antibodies (sc-166475) were both purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Mouse anti-His monoclonal antibody (M0812-3) was purchased from Hangzhou HuaAn Biotechnology Co., Ltd. (Hangzhou, China). Horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin G (IgG; SA00001-1) was purchased from Proteintech (Chicago, IL, USA). AA was purchased from Alfa Aesar (Ward Hill, MA, USA). All other chemicals and reagents used were of highest purity.

The soluble inclusion body proteins were applied to a Ni²⁺-NTA Superflow Cartridge (Qiagen) equilibrated with binding buffer. The column was next washed sequentially with binding buffer D followed by washing buffer (8 M urea, 20 mM sodium phosphate, pH 7.4, 500 mM NaCl, 0.1 mM PMSF, 1 mM β-mercaptoethanol and 40 mM imidazole) and then eluted with elution buffer (8 M urea, 20 mM sodium phosphate, pH 7.4, 500 mM NaCl, 0.1 mM PMSF, 1 mM β-mercaptoethanol and 500 mM imidazole). The purification of denatured trCOX-2 was monitored by analyzing aliquots of the collected samples using 12% SDS-PAGE and then stained with Coomassie Brilliant Blue R-250. The desired eluted proteins were refolded as previously described (26 ). Briefly, the purified denatured trCOX-2 products were diluted 1:10 in refolding buffer E (42 mM Tris-HCl, pH 8.0, 62 mM HEPES, 2.5 mM DTT, 0.1 mM CaCl₂, 0.5 M arginine) and slowly stirred on ice for 4 h to allow COX-2 renaturation to occur. The renatured trCOX-2 was stored at −80°C following determination of protein concentration using the Bradford assay.