Buffer Considerations when using Anion Exchange (FPLC)

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dinofernando
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Buffer Considerations when using Anion Exchange (FPLC)

Post by dinofernando » Tue Feb 23, 2010 12:01 pm

Hi Everyone!

Quick question about protein purification using Anion Exchange column, I'm a little new to the equiptmnt and protein purification. I have a HIS-tagged protein that has a pI above 7.5.

I'm using a 1M Tris, 50mM EDTA buffer at pH 7.5.

So that means in this buffer my protein will have a net positive charge (hence I cannot use anion exchange????)

So my question is what about the HIS tags, their negative can't I utilize them even though the protein is overall positive?

Thank you!
-Dinesh
Ph.D. Student
UOIT

dinofernando
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Post by dinofernando » Tue Feb 23, 2010 12:02 pm

Or am I able to dialize my protein into a more basic buffer?
-Dinesh
Ph.D. Student
UOIT

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JackBean
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Post by JackBean » Tue Feb 23, 2010 1:13 pm

The retention doesn't depend only on overall charge (pI), but also on local cationic/anionic patches, thus your protein can be retained even at pH 7.5
But even if not, the other proteins can be retained and thus your protein of interest purified. That's my case, I'm using High Q at pH 8.0
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

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