Hi,
It could help if you said what your ligand is.
Nadir
Pr. Nadir T. Mrabet
Structural& Molecular Biochemistry
Nutrigenex - INSERM U-954
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax: +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet<at> medecine.uhp-nancy.fr
On 08/10/2010 03:05, SIPPEL,KATHERINE H 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
