May be your complex components interact with column? I mean: you have calibration plot for column, right? Does the molecular mass of proteins calculated from elution volume equals to mass calculated by another method (like SDS-PAGE)?
2014/1/21 Keller, Jacob <kell...@janelia.hhmi.org> > Maybe there is something required for interaction that was in the buffer > used for the other binding studies, but not in your SEC buffer? > > > > JPK > > > > *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of > *David > Briggs > *Sent:* Tuesday, January 21, 2014 10:52 AM > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* [ccp4bb] Isolation of protein-protein complexes. > > > > 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 > -- Eugene Osipov Junior Research Scientist Laboratory of Enzyme Engineering A.N. Bach Institute of Biochemistry Russian Academy of Sciences Leninsky pr. 33, 119071 Moscow, Russia e-mail: e.m.osi...@gmail.com
