Especially if you're dealing with lysate, I suspect the best way to do it is with magnetic Ni beads that you lift up and out of the gunk, to help avoid false positives from aggregating stuff that SDS/urea/guan would all "elute".
But why do you want X to remain on the column/beads? Removing Y but not X probably requires knowing more about the nature of their interaction than you do at this point? (e.g. will it take high salt, pH change, low (1M) urea, mild detergents etc) ===================================== Phoebe A. Rice Dept. of Biochemistry & Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 http://www.rsc.org/shop/books/2008/9780854042722.asp ---- Original message ---- >Date: Thu, 23 Dec 2010 09:11:31 -0600 >From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> (on behalf of Jacob Keller ><j-kell...@fsm.northwestern.edu>) >Subject: [ccp4bb] SDS and IMAC >To: CCP4BB@JISCMAIL.AC.UK > >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 >*******************************************
