Purifying Protein from Inclusion Bodies
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Buffer A |
Buffer B |
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50 mM tris-HCl, pH 8.0 |
20 mM Na2HPO4, pH 7.2 |
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5 mM EDTA |
20 mM NaCl |
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10 mM NaCl |
5 mM EDTA |
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25% (w/v) sucrose |
1.Weigh the centrifuge bottle (w/cap)before adding culture media.You will need to know how many grams of cells you have before you start lysis.
2.Spin down cells fromlarge culture at 6000 rpm for 20 min.If you have a lot of media you can do multiple spins pouring out the supernatant after each spin.
3.When spinning is complete weigh the flask (w/cap)and the cell pellet.Subtract the total weight from the empty flask weight to determine how many grams of cells you have.
4.Add 3 ml of buffer A to the flask for each gram of cells and fully resuspend the cell pellet.
5.Transfer the suspended pellet to 50 ml conical centrifuge tubes.
6.Add enough 100 mM PMSF to make a 1 mM final solution (10μl 100 mM PMSF/ml of solution).
7.Add 16μl of 50mg/ml lysozyme per gram of cells and mix.
8.Place in 37℃ water bath until solution becomes viscous.
9.Sonicate the solution to chop up the DNA and reduce the viscosity.
10.Spin solution at 18,000 rpm for 30 min.Save supernatant.
11.Add 3 ml of buffer B to the tube for each gram of cells and fully resuspend the cell pellet.
12.Add the same amount of 100 mM PMSF as you did in step 6 and 10μl Triton X-100 per ml of solution and resuspend the pellet.
13.Spin at the highest speed possible (~20,000 rpm)for 20 min.Save supernatant.The pellet remaining will be the inclusion bodies.
14.Add 20 ml of 8 mM urea (add DTT to 8 mM urea if purifying proteins with cysteine residues)and dissolve the pellet.Heating in a bath (37-50℃)may facilitate the pellet dissolving.
15.Repeat step 14 until no more of the pellet dissolves.
16.Dialyze 8 M urea solutions in 4 L of a 50 mM tris-HCl buffer solution at pH 8.5 using 3.5 kD molecular weight cutoff dialysis tubing for 2 days.Repeat,dumping out buffer and replacing with fresh buffer.
17.Empty dialysis tubing into 50 ml conical centrifuge tubes and spin at 20,000 rpm for 30 min.Freeze supernant until HPLC purification.
18.Clean up and wash all centrifuge tubes and flasks that you used.
Next step is to HPLC purify the protein.Before this step the solutions MUST be filtered through a 0.2u or 0.45u filter.
