Protein Purification from inclusion bodies

Lars Komorowski larskomo at biochem.mu-luebeck.de
Tue Nov 17 03:03:39 EST 1998


I recommend differential centrifugation. This is how I do it:
1. Pellet the cells 4°C, 7000g, 20 min
2. Resuspend the pellet in 1/25 Vol. (of your culture volume)  of the buffer
of your choice, 1x PBS e.g.
3. Add 0.5 mg/ml lysozyme if you want -> wait for 20 min
4. Sonify
5. Pellet the IBs 4°C, ca. 7000g, 20 min (I use a Heraeus Labofuge GL at
5500 rpm)
6. Resuspend the pellet as before, the rest is membranes and cytosol
7. Sonify
8. Pellet the IBs 4°C, ca. 7000g, 30 min
9. Resuspend the pellet in 1/125 Vol. (of your culture volume)

If you want you can wash the IBs with low detergent concentrations, 10 mM
SB-12 e.g., (not recommended because it is difficult to remove detergent),
or with low denaturing reagent concentrations, 1M guanidinium or 2M urea
e.g., to remove contaminations





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