Hi Kushol, No. The void for the column is 8ml and the whole volume of the column is 24ml. You must be talking about a different column.
Zhen -----Original Message----- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Kushol Gupta Sent: Thursday, June 20, 2013 4:09 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Puzzling observation about size exclusion chromatography Isn't 18 mLs into a Superdex 200 10/300 column run out near where the 670kD marker is, just after the void at ~15 mLs? Zhen, did you mean ~500kD rather than 5kD?..... Kushol Kushol Gupta, Ph.D. Research Associate - Van Duyne Laboratory Perelman School of Medicine University of Pennsylvania kgu...@mail.med.upenn.edu 215-573-7260 / 267-259-0082 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 [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Patrick Loll [pat.l...@drexel.edu] Sent: 20 June 2013 20:39 To: CCP4BB@JISCMAIL.AC.UK 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 > > > 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.=
