Use Urea - it does not interfere with gels etc. Additionally, you should consider covalent immobilization of protein X - using activated resins. Amine and carboxylic acid immobilization is common, however my all-time favorite is iodoacetamide-activated resin reacting with SH on the protein (if you have surface-exposed Cys - great, if you don't - just add one at a convenient spot, since your protein is recombinant anyway). While IMAC is a 'fairly strong' label, there will be cases where it leaches off, potentially complicating your analysis. Also you cannot reliably use it in the presence of quite a few chemicals that may be necessary for maintaining the competent state of protein Y. Also covalent modification allows you to elute with a variety of reagents including things that are not compatible with IMAC (e.g. low pH).
May the Holiday Chipmunk regurgitate many presents under your Yuletide Shrub. Artem On Thu, Dec 23, 2010 at 9:11 AM, Jacob Keller < [email protected]> 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: [email protected] > ******************************************* >
