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:mfrank...@nysbc.org] Sent: Thursday, June 20, 2013 4:34 PM To: Zhang, Zhen Cc: CCP4BB@JISCMAIL.AC.UK 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, > > 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 > > > The information in this e-mail is intended only for the person to whom it is > addressed. If you believe this e-mail was sent to you in error and the e-mail > contains patient information, please contact the Partners Compliance HelpLine > at > http://www.partners.org/complianceline . If the e-mail was sent to you in > error > but does not contain patient information, please contact the sender and > properly > dispose of the e-mail. > > -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
