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
>>>
>
>
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