In my experience, either urea or guanidinium crashes out in gels. I can't remember--which one is it? I am thinking guanidinium. (If the answer to this email saves one grad student from the aggravation of such a phenomenon, it will have been worth it...)
JPK On Thu, Dec 23, 2010 at 9:31 AM, Preben Morth <j.p.mo...@ncmm.uio.no> wrote: > Hi Jacob > > Why not try with urea and > for this type of studies I would probably use batch with the IMAC resin and > not run the samples over a column. > > cheers > Preben > > On 23/12/2010, at 16.11, Jacob Keller wrote: > >> Dear Crystallographers, >> >> I am interested in doing a type of pull-down experiment by >> immobilizing protein X on IMAC resin, flowing a large volume of dilute >> lysate containing protein Y over it, then adding some concentrated >> agent (solid SDS perhaps) to some more of the same lysate, and running >> that over the column to elute protein Y off protein X, without eluting >> X off the column. I am afraid from past experience that SDS might >> knock X off the column, presumably depending on the concentration. I >> do not care about the folding state of Y--I will just be running a >> PAGE gel anyway. Does anyone know either what is the minimal >> concentration of SDS for robustly unfolding proteins/breaking up >> interactions (and whether that concentration is safe for IMAC), or >> what would be a good alternative agent to do the same? >> >> Thanks in advance for your help, >> >> Jacob Keller >> >> ******************************************* >> Jacob Pearson Keller >> Northwestern University >> Medical Scientist Training Program >> cel: 773.608.9185 >> email: j-kell...@northwestern.edu >> ******************************************* > > J. Preben Morth, Ph.D > Group Leader > Membrane Transport Group > Nordic EMBL Partnership > Centre for Molecular Medicine Norway (NCMM) > University of Oslo > P.O.Box 1137 Blindern > 0318 Oslo, Norway > > Email: j.p.mo...@ncmm.uio.no > Tel: +47 2284 0794 > > http://www.ncmm.uio.no/research/ncmm-embl-group-leaders/ > > -- ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *******************************************
