Editing Zinc Finger Nuclease Purification (section)

== '''Purify from Inclusion body'''  ==

1. Inoculate 500 ml of LB medium with 5 ml of the overnight culture.<br>2. Incubate cells at 37 °C with shaking until the OD600 reached 0.4.<br>3. Incubate at room temperature with shaking until the OD600 reaches 0.9. <br>4. Add 0.4 mM IPTG and 4 mM arabinose and incubate for 16 h.<br>5. Spin down cells by centrifugation.<br>6. Resuspend the pellet in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% Triton X-100, pH 8.0). <br>7. Lyse cells by sonication.<br>8. Spin at 10,000 rpm for 20 min at 4 °C.<br>9. Wash inclusion bodies with lysis buffer.<br>10. Spin at 10,000 rpm for 20 min at 4 °C.<br>11. Repeat step 9 to 10 twice.<br>12. Wash pellet with cold water then PBS and each time spin down pellet.<br>13. Incubate pellet with solubilization buffer (PBS, 500 mM l-arginine, 90 μM ZnCl2, 1 mM MgCl2, pH 7.4) for 3 h at 4 °C.<br>14. Spin for 10 min at 10000 rpm.<br>15. Collect supernatant and purify protein using Ni beads.<br> 

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