September 23, 2019, 04:16:45 PM
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Topic: How to Separate Proteins with Similar Charge using Ion Exchange Chromatography  (Read 1624 times)

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Offline moondaughter

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I have a protein with a pI of 4.5 and another with a pI of 4.8.

This and the molecular weight of both of the proteins, which are in close range, as well as their Km values are the only things that I know about these proteins. (They are actually isozymes which explains the similarities)

I remember something my TA said something about using a pH gradient. He said that the isozymes should come out in two different peaks on an elution profile.

But I'm confused about whether I should use an anion or cation exchange column? Does it matter which one I use? Does this also mean that the buffer that I suspend my protein in has to be greater than the pI for the anion and the opposite for the cation?

If someone could just help me get through the procedure of the column that would be greatly appreciated!

Thanks for all *delete me*

Offline Babcock_Hall

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Either CEX or AEX should work, and one could envision either increasing or decreasing the pH over time.  The order of elution would be dependent on both choices.  At pH values above the pI, the protein will have a net negative charge.  At pH values below the pI, the protein will have a net positive charge.

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