Dear Reza, Regarding the molecular weights (60 to 360 kDa), the low salt concentration seems to drive the assembly of an hexamer. Salt concentration could have a drastic effect on protein oligomerization. If your A280 profile of the 360 kDa peak looks good (symmetric and in the resolution limits), it is very likely to be an "hexamerization"! As Nicolas FOOS said, you could confirm it using DLS or SEC-MALS (even better). Best wishes, Nicolas
*Nicolas RICHET, Ph. D.Post-Doctoral ResearcherUniversity College Cork* *School of Microbiology* *Food Science and Technology Building* *College Road* *Cork City* *IRELAND* *Mobile: +353 (0)838385151* 2017-01-12 9:26 GMT+00:00 Nicolas FOOS <nicolas.f...@esrf.fr>: > Dear Reza, > > > in the past I had work with protein able to oligomerize reversibly but > when oligomerization happened, even if I was able to separate and obtain > monomeric protein, > > protein was not in a good condition. > > > Have you try to characterize the two different states by DLS ? To > discriminate "interaction with resin" that you suspect from oligomerization. > > Nicolas > > > Nicolas Foos > PhD > Structural Biology Group > European Synchrotron Radiation Facility (E.S.R.F) > 71, avenue des Martyrs > CS 40220 > 38043 GRENOBLE Cedex 9+33 (0)6 76 88 14 87 <+33%206%2076%2088%2014%2087>+33 > (0)4 76 88 45 19 <+33%204%2076%2088%2045%2019> > > On 12/01/2017 01:50, Christopher Colbert wrote: > > What's your monomeric molecular weight? Increased salt concentration can > easily drive oligomerization. > > What is your evidence that it interacts with the resin? > > Cheers, > > Chris > > -- > Christopher L. Colbert, Ph.D. > Associate Professor > Department of Chemistry and Biochemistry > North Dakota State University > P.O. Box 6050 Dept. 2710 > Fargo, ND 58108-6050 > PH: (701) 231-7946 > FAX: (701) 231-8324 > > From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Reza > Khayat <rkha...@ccny.cuny.edu> > Reply-To: Reza Khayat <rkha...@ccny.cuny.edu> > Date: Wednesday, January 11, 2017 6:42 PM > To: "CCP4BB@JISCMAIL.AC.UK" <CCP4BB@JISCMAIL.AC.UK> > Subject: Re: [ccp4bb] Off-topic question about SEC > > All these make sense. Protein is very strange cause it goes from 60kDa > (globular) to an apparent 360kDa. Process is reversible too. > > > Reza Khayat, PhD > Assistant Professor > City College of New York > Department of Chemistry > New York, NY 10031 > ------------------------------ > *From:* CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Keller, > Jacob <kell...@janelia.hhmi.org> > *Sent:* Wednesday, January 11, 2017 7:39 PM > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* Re: [ccp4bb] Off-topic question about SEC > > > Yes if it either > > > > A) oligomerizes > > B) significantly changes shape > > C) aggregates reversibly > > > > On option B: Lower NaCl could make the protein “appear” bigger by > unfolding it a bit; hydrophobic interactions should be weaker in lower NaCl. > > > > JPK > > > > > > > > > > > > Artem > > www.harkerbio.com > > "where wild SEC columns roam free" > > > > On Jan 11, 2017 7:22 PM, "Reza Khayat" <rkha...@ccny.cuny.edu> wrote: > > Hi, > > > > Sorry for the off-topic question. Can a protein in lower [NaC] run faster > on a SEC than at higher [NaCl] (i.e. elute at an earlier volume)? The > protein elutes well within the resolution limits of the SEC > with a symmetric gaussian A280 profile. I know that at lower [NaCl] the > protein can elute later because it may interact with the matrix. Thanks. > > > > Best wishes, > Reza > > > > Reza Khayat, PhD > > Assistant Professor > > City College of New York > > Department of Chemistry > > New York, NY 10031 > > > > >
