Dear James,
I don't have a reference, but in my experience, simple pull-downs using coomassie staining will only work when the binding is better than a micromolar (although in essence the success really depends on the binding kinetics: slow dissociaters can be detected even when the overall affinity is low).
You can ramp up the sensitivity a few orders of magnitude by more sensitive detection methods (simple Western blot), but then the risk for false positives becomes higher. Why not try a quantitative method (ITC, SPR) provided you have access of course.
sincerely,
Filip Van Petegem
On Tue, Sep 21, 2010 at 12:01 PM, James Qiu
<[log in to unmask]> wrote:
Dear All,
Firstly, sorry for the non-crystallography question but I am trying to do a pulldown assay using Cobalt-NTA resin between two DNA replication proteins one of which contains a C-terminal 6x his tag. According to previous genetic studies, these two proteins are involved in replication and many believe there is a complex formed between the two. My question is does anyone know if there is a range of affinities for which a pull down assay is appropriate? And if so is there a reference one could recommend?
Secondly, if there is a weak interaction between two proteins, for example millimolar affinity, does that decrease the chances of co-crystallizing the two interacting?
Thank you in advance,
Sincerely,
James Qiu
--
Filip Van Petegem, PhD
Assistant Professor
The University of British Columbia
Dept. of Biochemistry and Molecular Biology
2350 Health Sciences Mall - Rm 2.356
Vancouver, V6T 1Z3
phone: +1 604 827 4267
email:
[log in to unmask]http://crg.ubc.ca/VanPetegem/
