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/
