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'
Case 932, 163 Avenue de Luminy, 13288 Marseille cedex 9, France
Tel: +33 491 82 55 93
Fax: +33 491 26 67 20
e-mail: philippe.le...@afmb.univ-mrs.fr
