Kd is crucial. If this is something like streptavidin and biotin, there is no way to separate them without denature the protein. You can try varying pH, binding it to ion exchange column and then washing it with large volume of buffer, as mentioned in MBP manual from NEB, or running it through a hydrophobic column after treating the protein with really high salt. The last one worked for one of my colleagues. Although I still doubt you can completely get rid of the ligands.
Nian Huang, Ph,D. Dept. of Biochemistry UT Southwestern Medical Center Dallas, TX 75390 On Thu, Oct 7, 2010 at 8:05 PM, SIPPEL,KATHERINE H <ksip...@ufl.edu> wrote: > Hi all, > > I am working with a substrate binding protein. The protein scavenges its > endogenous ligand out of the E. coli used for expression. I need to get this > ligand out for both crystallographic and kinetic studies. I have tried > denaturing in urea and refolding the protein with limited success. It > refolds properly according to the CD spectra but it some how manages to hold > on to trace amounts of ligand despite serial dialysis (500ml to 5ml of > sample) in 8M, 6M, 4M, 2M 1M urea followed by 50mM Tris. I also have a > homolog that abjectly refuses to refold in either urea or guanidine, though > it does turn the dialysis tubing into a lovely snow globe. There are > alternative methods of performing the kinetics, but those will require > destroying the protein which doesn't help on the crystallography front. > > I was wondering if any of you out there had experience successfully removing > very tightly bound ligands by an alternative method. I didn't see any > mention on the subject in the archives. I had hoped you might be able to > point me in the right direction. > > Thanks for your time, > > Katherine > > Ph. D. candidate > Department of Biochemistry and Molecular Biology > College of Medicine > University of Florida >
