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
