Purification of 6xHis-Tagged Proteins

2003-01-02 (from Chris Udell)

 

Where (Sample) is noted, take an aliquot of the material and boil in SDS-PAGE sample buffer for running on a gel.

 

1)                  Inoculate single colony of BL21(DE3) carrying desired plasmid into 20 ml TB + ampicillin (100 mg/ml) and culture overnight at 37C

 

2)                  Dilute sufficient amount of overnight culture into 1 L TB + ampicillin to give OD600 = 0.2 and culture at 37C for 1 hour (OD600 approx. 0.5-1.0)

 

3)                  Add IPTG to 0.1-0.4 mM and culture 4-6 hours at 37C

 

4)                  Pellet cells at 6,000 rpm for 5 mins in JA-10 or JA-14 and store at -20C

 

5)                  Resuspend cells in 50 ml lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) to which you have added 0.1 % (v/v) Triton X-100, 10 mg/ml RNase A (10 mg/ml stock), 5 mg/ml DNase I (5 mg/ml stock), 1 mM MgCl2 (1 M stock), 1 mM MnCl2 (1 M stock), and 1 mg/ml lysozyme (sample)

 

6)                  Incubate on ice 30 mins; sonicate (10 sec pulses with 30 sec intervals on ice) until becomes partially clarified

 

7)                  Centrifuge at 15,000 rpm in JA-20 for 20 mins (sample)

 

8)                  Add 5 ml Ni-NTA Agarose (pre-washed with lysis buffer) to supernatant and incubate at 4C with mixing for 30 mins

 

9)                  Pour mixture into column and let flow-through drain out (sample)

 

10)              Wash column with 2x50 ml of wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0)

 

11)              Elute protein in five 5 ml fractions with elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0) Do this step by adding 5 ml elution buffer to column, mix thoroughly, and let 5 ml drain from column. Repeat 4 more times.

 

12)              Analyse 3 samples and 5 fractions by SDS-PAGE

 

13)              Pool desired fractions and dialyse against buffer suitable for anion exchange chromatography on FPLC