Ni nta affinity chromatography resin

Bacteria expressing PNDD0, PNDD1 or PNDD2 were resuspended in 3 mL of lysis buffer (Tris 50 mM pH 8, NaCl 170 mM, imidazole 20 mM, urea 6 M, NP40 0.5% v/v) for a pellet corresponding to 60 mL bacterial culture. The solution was homogenized and sonicated. Insoluble material was discarded by centrifugation for 30 min at 16,000 g at 4 °C. 2 ml of packed Ni–NTA affinity chromatography resin (Qiagen, USA) was calibrated with 10 volumes of lysis buffer, before being incubated with soluble PNDD0, PNDD1 or PNDD2 overnight on rotor at 4 °C. Resin was then washed with 10 volumes of washing buffer (Tris 50 mMpH 8, NaCl 170 mM, imidazole 40 mM, urea 6 M, NP40 0.5% v/v). PNDD0, PNDD1 or PNDD2 were eluted twice with 1 volume of elution buffer (Tris 50 mM pH 8, NaCl 170 mM, imidazole 1 M, urea 6 M, NP40 0.5% v/v). Purified protein elute was injected in Slide-A-lyzer Dialysis Cassette 20,000 MWCO (Thermo Scientific) and incubated in 3 baths of 100 volumes of PBS for 21 h at 4 °C. Dialysed elute is retrieved from the cassette and tested for solubility by spinning down for 30 min at 15,000 g 4 °C. The supernatant is retrieved and stored at -80 °C. The pellet is resuspended in the same volume with lysis, washing or elution buffer. A sample is taken from each fraction to compare the amount of purified protein.