My protein (64kDa) expressed in insoluble inclusion bodies. I tried to purify it by ion-exchange and affinity but unable to purify protein. So, want to try ammonium sulfate precipitation of dissolved protein in 8M Urea. Will it work?
It will work. But, ammonium sulphate does not precipitate target proteins selectively. It often co-precipitates other highly expressed contaminant proteins. You may need to optimize the concentration of ammonium sulphate for selective salting-out of your target proteins.
It is also worth to try other precipitating agents, such as sodium sulphate, PEG, etc...
In the presence of urea, the solubility of your protein will be high in solution. So you need to consider protein concentration and concentration of precipitating agents for selective salting-out of your target proteins.
Yes everything is possible but the question is: what for? In my mind few things can be done with such preparation.
Hi Vikas,
I have experienced a number of proteins that refuse to bind to affinity resin - sometimes the problem has been solved by changing conditions, and other times by modifying the expression construct to use a tag that is more tolerant of the conditions necessary to extract the protein from cells and isolate from lysate in the first instance.
Can you be more descriptive of what it is you have tried (protein pI, the affinity tags in the sequence, affinity resin used, buffer conditions in your binding attempts etc.)? This will allow those of us reading your post to offer more helpful advice based specifically on the evidence presented. Careful selection of the lysis buffer composition may also give conditions that facilitate isolation of your protein and removal of contaminants.
One idea that comes to mind - 8M Urea dissolves almost everything so this will be a very crude solution with significant contamination. If you firstly take your pelleted cells and lyse with a plain saline buffer to remove the easily soluble proteins, then spin at top speed (ie. 48,000x g for 30 minutes), you can remove the soluble proteins in the supernatant. Repeat this, collecting the supernatant each time. Then, use buffer containing 1M urea to extract proteins soluble in 1M, then 2M, then increasing amounts of urea until you attain 8M urea, each time collecting the newly-solubilised proteins and separating from pelleted material. When you have reached 8M urea, perform SDS-PAGE of the collected supernatants and identify which fraction contains your protein of interest and how clean it is.
This means that if your protein is only soluble in 8M urea, you have a chance to remove many impurities that are soluble in lower levels.
Providing the NaCl concentration is low (or 0mM) , ion exchange can be performed in 8M urea, with elution by addition of NaCl or change of pH, all still with urea present.
This is only a starting point of the discussion - if you can reply with more information about your existing conditions we should be able to work this out. Best wishes, Robin
@ Robin Dickinson,
Sir, Since I am getting my protein in insoluble form I am using denaturing condition to purify the protein of interest.
Affinity Chromatography
My protein has pI of 6.5, having N-terminal His-tag. I have tried binding at pH range- 7-8 with 20mM Imidazole, with additives NaCl upto 500mM, Glyceral upto 5%; and my protein start eluting in 50mM Imidazole. While increasing percentage of additive adversely affect binding of protein to Ni-NTA resin.
Ion-Exchange Chromatography
I have also tried anion exchange chromatography with DEAE spharose. I have tried binding pH range from 8-11. Washing with reduced pH and low concentration of NaCl (25mM) and eluted with 50mM NaCl or at reduce pH 7.0 with 25mM NaCl.
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