Hi all, Thanks for the incredibly fast and helpful responses, both on and off list.
To provide some further information. I have tried the SEC with several buffers, including buffers identical to the SPR and DPI experiments. To-date all SEC and SPR experiments have been conducted at room temperature - so suggestions to repeat SEC in the cold room are useful and might yield complex. I am also fortunate enough that only one protein is (His) tagged, so co-purification on an IMAC resin is also something that I shall try. Regarding the SPR, many have highlighted that the most important parameter vis complex stability is Koff - I don't have those numbers right now (on the train) but from memory the off rate is slow, and the interaction does not appear to be the described fast-on/fast-off non-specific interaction. I always run a blank for all my SPR experiments and no non-specific interactions with the blank surface were seen Thanks once again everyone, Dave On 21 Jan 2014 15:51, "David Briggs" <drdavidcbri...@gmail.com> wrote: > 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 >
