Hello everybody,

I need your suggestions for the protocol that i have used before.

My protein size is ~12 kDA, it is N-term GST, C-term His tagged. I planned to use Ni-NTA. As you know, there can be two strategies:

1) Binding to Ni-NTA, elution, getting the purified protein. Doing thrombin digestion, and then rebinding the digested protein to Ni-NTA and finally elution.

2) Binding to Ni-NTA, doing a on column cleavage, and finally elution.

I tried using the first strategy, and found the amount of protein I get in the final elute is too less.

Whereas, when I use the second strategy, starting with close to 100 ml of induced culture, what I get it about 300 ng per ul of purified protein (close to 3 ml). I dont know if that is justified as concentration.

I have put down the procedure below and my doubts about specific steps. (in italics)

Procedure:

1) Resuspend the cells in about 6 ml of buffer. (100 ml pellet)

Composition: 50mM NaH2PO4, 300mM NaCl, pH 7.6

Add: 1 mM PMSF, 10 mM Imidazole

2) Add 0.1 volumes of 10 mg per ml lyzozyme. Incubate on ice for about 10mins.

3) Sonicate the cells at 32% amplititude for 10 secs at intervals of 30 sec for 40 secs on ice.

4) Spin down at max. speed for 10 mins.

5) Collect the supernatant. Keep about 10 ul of supernatant for SDS PAGE analysis.

Resuspend pellet in another 1ml lysis buffer and keep sample of 40ul for PAGE-SDS.

Column preparation and equilibration:

1) Take about 3.2 ml of slurry (Ni-NTA) after mixing it properly. (for 100 ml pellet)

( I don’t if I am taking too high quantity of Ni-NTA, I use the one from Qiagen, which typically has binding capacity of 50 mg per ml)

2) Wash the slurry with about 10 ml of water twice. Spin down at about 3000 rpm for 5 min.

3) Wash with about 10 ml of binding buffer (Buffer 50mM NaH2PO4, 300mM NaCl, pH 7.6) twice.

4)Add the supernatant to the equilibrated beads and allow for overnight binding at 4 degrees.

5) Spin down. Collect the flow through.

6) Wash with about 10 ml of chilled binding buffer (plus, 10% glycerol). Collect the flow through. Repeat step.

7) Wash with about 10 ml of chilled binding buffer (20 mM imidazole, 400 mM NaCl, 10% glycerol). Collect the flow through. Repeat step.

8) Wash with about 10 ml of chilled binding buffer (40 mM imidazole, 500 mM NaCl, 10% glycerol). Collect the flow through. Repeat step.

On column cleavage:

1) Wash with 9 ml of thrombin washing buffer.

(20 mM Tris HCl, 200 mM NaCl, pH 7.5)

2) Add 1 volume thrombin buffer plus 0.1 volume of 10X thrombin cleavage buffer (1.5 M Tris HCl, 1.5 M NaCl, 25 mM CaCl2 pH 8.4; supplied readymade) plus thrombin. (Initially, we were using 7 units per 100 ug of protein, Thrombin: 1.2 units per ul).

Not sure about how much thrombin to use. Is there any direct correlation between units of thrombin and amount of protein?

25 ml pellet: 900 ul of Thrombin buffer, 100 ul of 10X thrombin cleavage buffer, 23.3 ul (28 units) of thrombin. (Considering about 400 ug of total protein)

100 ml pellet: 1700 of Thrombin buffer, 200ul of 10X thrombin cleavage buffer

70 ul (84 units) of thrombin (Considering about 1200 ug of total protein)

3)Resuspend the beads. Incubate at 4 degrees overnight.

4) After incubation, collect the flow through.

5)Wash with about 10 ml of chilled binding buffer (20 mM imidazole, 400 mM NaCl, 10% glycerol). Collect the flow through. Repeat step.

6) Wash with about 10 ml of chilled binding buffer (40 mM imidazole, 500 mM NaCl, 10% glycerol). Collect the flow through. Repeat step.

7) Elute with upto 3 ml, with 250 mM Imidazole (plus 400 mM NaCl) in binding buffer.

( I am not really sure, as to much elution volume, I should use when I elute in 3 ml, I typically get 300 ng per ul of protein)

8) Bradfords for protein estimation and ELISA/Western blotting (0.2/0.45 u membrane, depending on protein size) to confirm protein.

Eagerly waiting for your comments...

Thank you, and regards,

Kaushik