I have two proteins which have almost the same M.Wt. They also have isoelectric points (7.1 and 7.75). Can I separate them with Ion exchange chromatography(IEC)? What is the PI range by which Ion exchange chromatography separates proteins?
Any help would appreciate.
Thanks
Yes you can separate the two proteins by IEC. provided you choose ampholites in the appropriate range eg ph 6.8 to 7.8. An alternative could be to try DEAE Cellulose chromatography .Equilibrate column at pH 6.8 in HEPES buffer and elute with a NaCl gradient. Good Luck
Sure you can separate them. Considering their MW you may chose gel filtration chromatography (size exclusion chromatography). Also DEAE Sepharose and Q Sepharose can be used by performing column chromatography, preequilibrating the column with suitable buffers at appropriate pH values. Good luck
Ion exchange chromatogrphy columns bind proteins with a negative net-charge. You could affect the net-charge of your protein by vary the pH of your buffer. At a pH above its isoelectric point, your protein has a negative net charge and will bind to an anion exchanger.
I think a Q-sepharose column has a better resolution but nevertheless you can try a DEAE sepharose as well. With a longer gradient (~20 cv) you have a better chance to separate both proteins.
Good luck
It will be quite challenging to separate these proteins based on their pIs, provided that these are experimental pI values and not theoretical ones. Check their hydrophobicity pattern, using one of the tools in Expasy. if they are different, you can use hydrophobic columns, such as Butyl-Sepharose column from GE-healthcare.
good luck!
Since the MW of the two proteins are close, they would not get separated by gel filtration. It will be tough to obtain complete/ separation by IEC as pointed out in the previous answer. Are the proteins very different ? or are they very close in terms of amino acid sequence? If they are different, then you may have some luck with HIC (hydrophobic columns, like butyl), as pointed out in the previous post.
I already recommended trying isoelectrofocusing with ampholites in the range ph6.8 -8.0 or thereabouts. A cation exchanger like DEAE-cellulose equilibrated in HEPES buffer at pH 6.8 should be able to bind both proteins but release them at different NaCl concentrations.
Since IEX chromatography basically work on the surface pI of protein therefore it is important to note that both protein should have different surface pI (do not used theoretical pI).
Used CEX and performed the elution ether by using pH gradient or by chromatofocusing.
Used 20mM NaH2PO4 as binding buffer (ph 4.7) and 20 mM Na2HPO4 as elution buffer (pH ≈9.2) for cation exchange.
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