Dear Priya,
How long did you soak your crystals?
I had a similar problem. When I tried to co-crystallize my protein with
its substrate (5 mM) I never got any substrate bound. I tried soaking
with higher concentration (up to 50 mM) and this didn't work either. The
crystals were infinitely stable in the soaking solution but even after 72
hours there was no substrate bound. When I was almost giving up I tried
one last crystal which was in the soaking solution for a long time. I mean
a very long, several weeks or so. In this crystal the substrate was bound
by the protein. I don't really know why the binding kinetics was so slow.
The enzyme's affinity for the substrate was quite high and I didn't expect
an unusually long soaking time.
Maybe a very long soaking time could also help in your case?
Joern
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Priya Mudgal
Sent: Thursday, November 27, 2008 11:09 PM
To: [email protected]
Subject: [ccp4bb] Co-crystallization
Dear All,
I am trying to co-crystallize a protein with substrate/substrate analogue
and for some reason it is not working. I have tried to co-crystallize
with upto 2 mM substrate and it does not bind. The protein precipitates
out if I go higher concentration. Soaking does not work either. Is
there any suggestion that I should try before I give up on this.
Thanks a lot.
Priya
--
Priya Mudgal
PhD Student
Duke University
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Address:
Joern Krausze
University of Leipzig
Centre for Biotechnology and Biomedicine
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eMail: [EMAIL PROTECTED]
Phone: +49 (0)341 9731312
Fax: +49 (0)341 9731319
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