Dear Dave,
Indeed, an interaction in the nM range is strong, but its dynamic is
important. If the SPR sensorgram, if published, looks like a 'crenel',
this indicates high association/dissociation rates. In that case, your
complex can dissociate during the GF run.
You can try crystallisation at 4°C in order to lower the dynamic of the
interaction, this worked for me...
Good luck.
Philippe
Le 21/01/14 16:51, David Briggs a écrit :
> Dear all,
>
> sorry for the slightly off topic post,
>
> I have 2 proteins that have been shown to interact, by multiple
> groups, and by multiple techniques - namely ELISA, SPR and DPI.
>
> The Kd of the interaction as determined by SPR is on the order of 1 nM.
>
> I would very much like to crystallise this protein-protein complex,
> and as a first step I attempted to purify the complex by mixing the
> two proteins (same protein preps and same buffers as the SPR
> experiment) and then running them down a gel filtration column
> (Superose 6 - predicted size of the complex is ~500kDa).
>
> Somewhat irritatingly the two proteins separate beautifully on the
> column into two distinct peaks. There is no trace of complex formation
> when the peaks are analysed by SDS-PAGE.
>
> As far as I am aware, two proteins that interact this strongly should
> remain associated during gel filtration, and I was wondering if anyone
> else has encountered anything similar in the past, and if they managed
> to resolve the problem, how they went about it?
>
> Cheers in advance,
>
> Dave
> ============================
> David C. Briggs PhD
> http://about.me/david_briggs
--
Philippe Leone
AFMB-UMR7257
Team 'Structural Immunology'
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