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Protein purification using Ni-NTA resin

Proteins with a 6xHis tag can be purified through Nichel nitrilotriacetic (Ni-NTA) resin. In our laboratory, we purify the recombinant extradomain B of fibronectin (ED-B) through this technology. ED-B is a 91 aminoacids protein and it is sub-cloned in pQ12 vector, which appends a 6xHis affinity tag at the C-terminus.
Bacteria transformed with pQE12 are grown in 2XTY medium containing 0.1% glucose and 100 µg/ml ampicillin at 37 °C. At OD600 = 0.8, cells are induced by addition of IPTG and left at 25 °C overnight. The suspension is then centrifuged (30 min., 7500 rpm) and the pellet resuspended in Buffer A (20 mM imidazol, 0.25M NaCl, 10 mg/ml pefabloc (Boehringer Mannheim), PBS) and the cell suspension sonicated 3 X 1 min. using a XL ultrasonic processor. The cells are pelleted by centrifugation at 17000rpm for 20 min, using a Sorvall centrifuge equipped with an SS34 rotor. The supernatant is loaded on a Ni-NTA-Agarose column (2.5 ml per 800 ml initial culture broth), washed with Buffer A and eluted with Buffer B (200 mM imidazol in PBS). Fractions are dialysed overnight against PBS and frozen in liquid nitrogen.


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