[박상규] p43 purification

[Anonymous]

Buffers for p43 purification

 

Column Binding buffer (500ml)

20 mM KH2PO4, 500mM NaCl, pH 7.8, 2mM b-mercaptoethannol

 

Column Wash buffer 1(500ml)

20 mM KH2PO4, 500mM NaCl, 10% glycerol, pH 6.0, 2mM b-mercaptoethannol

 

Column Washbuffer 2(500ml)

20 mM KH2PO4, 500mM NaCl, 10% glycerol, pH 5.2, 2mM b-mercaptoethannol

 

column wash buffer 3 (500ml)

20 mM KH2PO4, 500mM NaCl, 10% glycerol, pH 6.0, 2mM b-mercaptoethannol + 100mM Imidazole

 

3M imidazole in D.W

 

Elution buffer

Dilute 3M imidazole to 300mM with column wash buffer 1

 

1X PBS containing 20% glycerol for dialysis

è DW for buffer should be 3rd. Solution for purification should be sterilized by filtration of 0.2um membrane and adjust pH with HCl, not dibasic phosphate. Mercaptoethanol should be added just before use.

 

Column: Ni++ resin 4ml

 

 

 

Procedure (500ml culture)

 

1. Resuspend the cell pellet with column binding buffer with 5ml/g(wet weight)

2. Sonicate the cells in column binding buffer for 20min with 50% power (1sec pulse, 2sec interval).

3. Centrifuge the lysate for 40 min at 26,000g at 4 degree

4. Harvest the supernatant and pass the solution with 0.4 syringe filter

5. Load the solution into the 4ml resin pre-equilibrated with 10 bed volume of column binding buffer.

6. Wash the column with 20 bed volume of column binding buffer.

7. Wash the column with 50 bed volume of column wash buffer 1.

8. Wash the column with 50 bed volume of column wash buffer 2.

9. Wash the column with 10 bed volume of column wash buffer 3.

10. Elute the p43 with elution buffer(2ml/frac, total 15 fraction)

11. Run the SDS-PAGE and check the protein purity and fraction

12. Dialyze the protein against 1X PBS containing 20% glycerol(2 times, at least 4hrs per one time)

 

è Purity will be at least 95%

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