Thanks to everyone for replying. The consensus so far is the protein is interacting with the column and so eluted later than it is supposed to. I do have 150mM NaCl in the running buffer. The success of refolding is questioned by many, which is also my concern. The protein is heavily engineered and the antibody we used is a highly specific one targeting on a structural epitope on the wild type protein. The binding assay was done by Octet with 1mg/ml BSA in the buffer. The assay was negatively controlled by antibody with buffer and buffer with the refolded protein and was positively controlled with the wild type protein. This seems to me a strong evidence that the protein is well folded. Please point it out if this is a wrong assumption. I will try to find a place to run CD spectrum for double check.
I should have mentioned that the late elution only happens when refolding with Arginine. The protein was eluted close to expected trimer position if refolded in other buffer but the refolding efficiency is significantly worse than with Arginine (more than 90% stays in void without Arginine vs. no visible peak in void with Arginine). I cannot make up my mind to go with the much less efficient path yet. Any suggestions?
Zhen
-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of RHYS GRINTER
Sent: Thursday, June 20, 2013 3:47 PM
To: [log in to unmask]
Subject: Re: [ccp4bb] Puzzling observation about size exclusion chromatography
Hi Zhen,
I'm not sure that binding to a monoclonal antibody is good evidence that the protein is in a natively folded state. I would be suspicious of such a result as the protein could be improperly, which is causing it to interact with the column matrix. It could be useful to use some other techniques (Activity Assay, Circular Dichroism, DSC, Native Page etc. to validate the refolding).
Best,
Rhys
________________________________________
From: CCP4 bulletin board [[log in to unmask]] On Behalf Of Patrick Loll [[log in to unmask]]
Sent: 20 June 2013 20:39
To: [log in to unmask]
Subject: Re: [ccp4bb] Puzzling observation about size exclusion chromatography
If your protein elutes very late, that means it's binding to the column matrix (so all estimates of size go into the trash). Check to see that the ionic strength of buffer is reasonable (equivalent to, say, 150 mM NaCl). If so, then the only solution is to go to a different matrix type.
Pat
On 20 Jun 2013, at 3:09 PM, Zhang, Zhen wrote:
> Dear all,
>
> I just observed a puzzling phenomenon when purifying a refolded protein with size exclusion chromatography. The protein was solubilized by 8M Urea and refolded by dialysis against 500mM Arginine in PBS. The protein is 40KDal and is expected to be a trimer. The puzzling part is the protein after refolding always eluted at 18ml from the superdex S200 column (10/300), which is calculated to be 5KDal by standard. However, the fractions appear to be at 40KDal with SDS PAGE and the protein is functional in term of in vitro binding to the protein-specific monoclonal antibody. I could not explain the observation and I am wondering if anyone has the similar experience or has an opinion on this. Any comments are welcome.
>
> Thanks.
>
> Zhen
>
>
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