 |
 |
Hi Michael,
Thank you very much for your suggestions. I will rerun with both buffer conditions and report back later.
Thanks to everyone for your reply.
Zhen
From: CCP4 bulletin board [mailto:[log in to unmask]]
On Behalf Of R. M. Garavito
Sent: Friday, June 21, 2013 9:28 AM
To: [log in to unmask]
Subject: Re: [ccp4bb] Puzzling observation about size exclusion chromatography
Dear Zhen,
I should also point out that the statement Matt made ("Superdex is known to have some ion-exchange characteristics, so that it can weakly interact with some proteins.") is not completely correct. Superdex and all chromatographic media
made from carbohydrates (dextran, agarose, etc.) are also quite hydrophobic (which is surprising to some). The observation Zhen made is the classic behavior of hydrophobic interaction with the gel filtration media, which led to the development of hydrophobic
interaction chromatographic (HIC). For HIC, you load the protein in high salt, and elute with low salt or a chaotrope (LiCl).
So when you redo you experiment, Zhen, try with AND without salt. As Matt said, if it is an ion-exchange interaction, extra salt will make it elute more normally. But if it gets worse, then the extra salt is increasing the hydrophobic
interactions, and you should run the column in lower salt (~50 mM NaCl) or with a bit of detergent. Given that you are refolding your protein, a partially folded protein may have have more hydrophobic patches. As my lab is routinely refolding our target proteins,
we are always watching for this behavior, including on our analytical Superdex 200 10/300 columns, which is one of our favorites. Excessive hydrophobic interactions can also lead to clogged columns, which is not what you want for this expensive column.
Good luck,
Michael
****************************************************************
R. Michael Garavito, Ph.D.
Professor of Biochemistry & Molecular Biology
603 Wilson Rd., Rm. 513
Michigan State University
East Lansing, MI 48824-1319
Office: (517) 355-9724 Lab: (517) 353-9125
FAX: (517) 353-9334 Email: rm[log in to unmask]
****************************************************************
On Jun 20, 2013, at 5:38 PM, Zhang, Zhen wrote:
Hi Matthew,
Thanks a lot. That is a great idea. I will try the high salt and worry about the crystallization later.
Zhen
-----Original Message-----
From: Matthew Franklin [mailto:[log in to unmask]]
Sent: Thursday, June 20, 2013 4:34 PM
To: Zhang, Zhen
Cc: [log in to unmask]
Subject: Re: [ccp4bb] Puzzling observation about size exclusion chromatography
Hi Zhen -
Superdex is known to have some ion-exchange characteristics, so that it
can weakly interact with some proteins. This is why the manufacturer
recommends including a certain amount of salt in the running buffer. I
have had the same experience with a few proteins, including one that
came off the column well after the salt peak! (The protein was very
clean after this step; all other proteins had eluted earlier.)
As others have said, you can't rely on molecular weight calibrations in
this case, but this behavior alone is no reason to think that the
protein is misfolded or otherwise badly behaved. If you don't like the
late elution, try increasing the salt concentration of your running
buffer to 250 or even 500 mM. You'll probably need to exchange the
eluted protein back into a low-salt buffer for your next steps (e.g.
crystallization) if you do this.
- Matt
On 6/20/13 3:09 PM, Zhang, Zhen wrote:
Dear all,