Pierce centrifuge column

Large 250 mL cultures were grown to an OD₆₀₀ of 0.6 in Miller LB supplemented with 0.1 mg/mL ampicillin before initiating cold-shock on ice for 1 h. Cultures were then induced with a final concentration of 1 mM IPTG, followed by growth at 15 °C for 48 h. The cells were collected by centrifugation at 4000 × g and 4 °C for 20 min and resuspended in a lysis buffer of 50 mM sodium phosphate pH 8.0, 300 mM sodium chloride, 10 mM imidazole, and 0.05% (vol.) polyoxyethylene (20) sorbitan monolaurate (Tween-20). The cells were lysed, following which the insoluble material was removed by centrifugation at 17,000 × g and 4 °C for 20 min, and the supernatant was filtered by syringe through a 0.22 μm filter. The filtered crude protein was then incubated with 500 μL of Ni–NTA agarose (Qiagen, Valencia, California) per culture at 4 °C for 2 h, collected on a Pierce^™ Centrifuge Column (Life Technologies, Grand Island, New York) by gravity flow, and washed with 2 column volumes of a wash buffer of 50 mM sodium phosphate pH 8.0, 300 mM sodium chloride, and 20 mM imidazole. The protein was then eluted in 500 μL increments with a stepwise gradient of imidazole in 50 mM sodium phosphate pH 8.0, 300 mM sodium chloride (four column volumes were collected at each stepped concentration).