Login

Join for Free!
112318 members


Buffer Considerations when using Anion Exchange (FPLC)

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

Moderator: BioTeam

Buffer Considerations when using Anion Exchange (FPLC)

Postby 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
Garter
Garter
 
Posts: 14
Joined: Sat Aug 04, 2007 1:50 am
Location: Oshawa, Canada UOIT

Postby 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
dinofernando
Garter
Garter
 
Posts: 14
Joined: Sat Aug 04, 2007 1:50 am
Location: Oshawa, Canada UOIT

Postby 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.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5652
Joined: Mon Sep 14, 2009 7:12 pm



Return to Molecular Biology

Who is online

Users browsing this forum: No registered users and 0 guests

cron