Hey, hi guys. I had a problem in purifying protein from bacterial inclusion bodies. I used bugbuster solution and inclusion body solubilization reagent from thermo-scientific and then went for dialysis using tris buffer (PH 7.5). After dialysis my protein got precipitated. How can I resolve this problem. Is there any buffer to redissolve and further to get fruitful results. Please help me out. Thank you
There are many possibilities to explore. Your protein may just need to have a higher salt concentration to stay in solution at the particular pH you are using. Also, try using different pH dialysis. Your protein may be in a denatured state after solubilization from inclusion bodies and will require careful attention to refolding conditions. Keep the protein concentration as low as possible to minimize aggregation. You can concentrate it later. There are kits you can buy to test various renaturation conditions on a small scale. If your protein normally has disulfide bonds, you may need to use a redox system (e.g. reduced + oxidized glutathione) to help the correct disulfide bonds form during refolding. Or, if your protein normally has no disulfide bonds you may need to keep the protein reduced during refolding to prevent unnatural disulfide bonds from forming.
all the advices are good but I would add one: do all the processes (dyalisis, etc.) at low temperature!
@ Xabier@ i am doing at 4 degree temp.is that fine sir.
Thank you all for suggestions.
Protein purification from inclusion bodies is a bit difficult task. First of all protein expressing in inclusion bodies itself says its instability so u need to take utmost care. I would say eluting the protein from inclusion bodies using some non-ionic detergent would help. Similarly, you can solubilize the protein from inclusion bodies which is present in the pellet of the cells after lysis using 8M urea or 6M Guanidine hydrochloride and then refold the protein step wise into a more appropriate buffer taking in to consideration the pI of the protein and other charecters as stated by Adam. check these out..hope it help. all the best
Once precipitated,... no way back. Well depends on the type but usually a solvent will dissolve anything, or a buffer containing high detergent.
for inclusion bodies many chemicals such as 8 to 12 M Urea or Guanidine hydrochloride.
There are also kits sold by Fisher/Pierce for screening 96 conditions for re solubilization. Best wishes
- Badges
- Science method