HTN-GFP purification

1.  Grow E coli transformed with 1918 pT7A3-HC-HTN-sfGFP or 1005 pBad24-Tat-sfGFP in LB with antibiotics and 2% glucose.

2. At OD600 =0.6, induce with 0.15 mM ITPG (for 1918) at 30 oC o/n, or 0.2% arabinose (for 1005) at 30 oC o/n

3. Spin down, lyse cells using buffer A containing 0.1% Triton X-100.

4. Load to Ni column, wash with Buffer A.

5. Elute with Buffer B containing 10% glycerol.

6. Add 4 M NaCl to adjust to final conc of 2 M NaCl, Load to 10 ml MEP column. Wash with 50 mM Tris-HCl, pH 7.0, 0.5 M NaCl.

7. Elute with Buffer containing 50% Glycerol, 0.1 M Arginine, 50 mM Tris-HCl, pH 7.0, 0.1 M NaCl, 1 mM DTT.

8. Filter through 0.2 micro filter, aliquote and store at -80 oC.