Dear all,
I would like to ask what is the maximal achievable resolution of ion exchange chromatography in the matter of pI values. In particular I am wondering whether I will be able to separate proteins which have pI of 7, 7.5 and 8. Can you please share your experiences?
Best Regards,
Dawid
It's probably possible to separate all of those, but can be very difficult. You need to optimize a lot, and discover "tricks" exploiting features of the protein structure. The first thing to understand is that PI does not reflect the actual charge difference.
Only accessible surface charges count. Buried charges, or charges that form internal salt bridges don't matter. Your PI prediction software almost certainly missed that, so reality could be quite different from what it thinks it is.
There is going to be a pH to be found at which the actual charge difference is larger than the PI difference. At other pHs, the actual charge difference is going to be smaller. You can try predict that with varying success. It's best to test experimentally at which pH your resolution becomes best.
And finally, look at sub-structures. If one of the proteins has a local high density of acidic or basic charges, you're in luck. Those can function essentially like a poly-R tag -- the local structure that can interact will interact with the resin, while the rest of the protein points away from it. Then, only the charge of the substructure will matter.
John, thank you very much for your reply. It is very helpful. Can you recommend any good literature on ion exchange chromatography?
Dawid your protein PI is having 7-8 it means you can use at pH 4-6 as cation exchange like CM /SP and pH 9-10.5 as anionic exchange like Q/DEAE or capto series.
first you have to check protein is soluble at pH 4-6 or 9-10.5 depend on that you can design you experiment
Resolution you can achieve by salt gradient, if it is not resolve, use salt with different pH gradient.
separation depend on protein structure and hydrophobicity also some time required organic solvent. some time if you use small bead size resin separation will come like 15 or 30 u.
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