I would like to thank all of you for your replies. I very much
appreciate your time and I've got some great starting points to
try out. I will put together a recap of the off- and on-board
comments in the next day or so for archive posterity.
For those looking for more details...
The protein is specifically a monomeric substrate binding protein
(very roughly similar to MBP) and binds a water-soluble vitamin.
It's purified exclusively by ion exchange so no tags though it is
shy a bit of the N-terminal domain to remove a lipo- moiety. I
have no idea what the Kd is at the moment, that was what I was
hoping to calculate. I do have the bound structure, it is not
covalently attached to the protein. The interactions are mostly
hydrophobic with only a couple of hydrogen bonds.
Once again, thank you all,
Katherine
On Fri, Oct 8, 2010 at 12:20 PM, Joseph Ho
<j...@medusa.bioc.aecom.yu.edu> wrote:
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,
>
> J??rgen
>
> -
> J??rgen 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
>>>
>
>
--
SIPPEL,KATHERINE H
Ph. D. candidate
Department of Biochemistry and Molecular Biology
College of Medicine
University of Florida
