I want to purify my protein ( PI 7.6). When I used buffer with pH 8, my protein aggregates. What should the pH of the buffer for purifying my protein be?
Hi Ajit,
which pH is suitable depends pretty much on the purification method you want to use! Since you didn't write any details on that a helpful advice is not really possible. Not only the pH is important, salt and other aditives might influence the outcome a lot!
So which purification method do you want to use?
Best,
Kai
Hi! Try to purify in Hepes buffer pH 5.0. Use a CM Sepharose equilibred in the same buffer and detach with a solt gradient from 0 to 2 M NaCl. best regards
Hi Ajit,
which pH is suitable depends pretty much on the purification method you want to use! Since you didn't write any details on that a helpful advice is not really possible. Not only the pH is important, salt and other aditives might influence the outcome a lot!
So which purification method do you want to use?
Best,
Kai
Be careful, proteins are less soluble near their native pHi (and more soluble far this pH). If pHi 7.6 is the calculated pHi of your protein (is roughly the pHi of the denatured protein), this information have no significance for the purification of a native protein. Try very different buffers (Tris, MOPS, Hepes but also phosphate buffer) and in a large range of pH. If your protein is hydrophobic, try to add small amount (below the micellar concentration) of a non ionic weak detergent. Some times, the presence of high salt concentration (up to 0.5M) is required.
And remember, each protein is very different from another !
In general its a rule to purify the protein +/- 1 units of pI. If the pH is more than pI the protein is negatively charged and can be purified using Anion Exchangers (eg: DEAE) and if the pH is kept less than pI the protein is positively charged and can be purified using Cation Exchangers (eg: -SO3H groups). Therefore the pH of the buffer you are using should be atleast either 6.6 or 8.6. You can use Tris-HCl buffer in that case. However as pointed out by Albring, there are many other factors such as ionic groups of the buffer you use, its strength , the type of protein you are trying to purify (native or recombinant) , the additive required to maintain the correct folding of your protein which would further decide the type of buffer required for protein purification.....Hope this info helps you. Good luck, Deepti
Another helpful piece of information would be to know if your protein is a globular protein or membrane protein as this may play a more significant role in the reason for aggregate formation than the buffer pH. While your buffer pH may be suitable, poor buffer conditions (eg: need for micelles/bicelles, higher salinity, etc.) can result in protein aggregation.
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