Hey David, the 1 nM Kd by SPR sounds fishy to me - did you do these measurements yourself ? Unless this is an antibody-protein complex or nanobody-protein complex or protein-small molecule complex, the value is too low (for a normal PPI, I would expect >50 -500 nM). Possible explanation for the SPR result is non-specific binding of the ligand (your protein in solution) to the chip. Which would in turn also explain your negative result in SEC eventually (what SEC material did you use ? Is it at the pH negatively charged perhaps - like the SPR chip). Are you running the SEC in the same buffer as used in either ELISA, SPR or DPI ? Most likely the main difference between those experiments and yours is the temperature, as I assume you ran your SEC at 4˚C. Just a thought.
Jürgen On Jan 21, 2014, at 10:51 AM, David Briggs <drdavidcbri...@gmail.com<mailto: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 ...................... Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
