Dear Filip, Thank you for your advice, in my lab there is a discussion where a senior investigator who suggested that if you cannot see evidence of an interaction by coomassie then you have no chance of crystallizing the complex. I disagreed but I did not have a reference to support my stance. I agree with you about the binding kinetics, I suspect if there is a slow off rate there is a chance you can capture that complex. If there is a slow on rate and a fast off rate then you are dead in the water I would imagine. Once again thank you for your comments.
Sincerely, James On Sep 21, 2010, at 3:32 PM, Filip Van Petegem wrote: > 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 <james....@tufts.edu> 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: filip.vanpete...@gmail.com > http://crg.ubc.ca/VanPetegem/
