It is just like the regular dialysis but the deserved buffer contains charcol powder.
Meng-Chiao Joseph Ho > How do you do this ? > I have not heard of this, but I also never had to deal with getting rid of > a ligand. > However I would be interested to learn more about this method. > > Thanks, > > Jürgen > > - > Jürgen Bosch > Johns Hopkins Bloomberg School of Public Health > Department of Biochemistry & Molecular Biology > Johns Hopkins Malaria Research Institute > 615 North Wolfe Street, W8708 > Baltimore, MD 21205 > Phone: +1-410-614-4742 > Lab: +1-410-614-4894 > Fax: +1-410-955-3655 > http://web.mac.com/bosch_lab/ > > On Oct 8, 2010, at 11:01 AM, Joseph Ho wrote: > >> 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 >>> > >
