We often dialysis protein against charcoal to remove small molecules that tightly bind to protein.
Meng-Chiao Joseph Ho, PhD Department of Biochemistry Albert Einstein College of Medicine > 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 >
