This doesn't directly address your question, but since the subject of analyzing protein-protein interactions with gel filtration is raised on this bb occasionally, I thought I would mention that there are cases in which conventional gel filtration chromatography fails to provide evidence of a known protein:protein interaction. In such cases, the Hummel-Dreyer gel filtration method is sometimes used. It involves supplementing the running buffer with one of the proteins, so you need a lot of protein. Here are two references:
Proc. Natl. Acad. Sci. USA 88 (1991) Biochemisrry (1993) 32, 11124-11131 On 11/19/10 6:58 AM, "Sebastiano Pasqualato" <sebastiano.pasqual...@ifom-ieo-campus.it> wrote: Hi all, I have a crystallographical/biochemical problem, and maybe some of you guys can help me out. We have recently crystallized a protein:protein complex, whose Kd has been measured being ca. 10 uM (both by fluorescence polarization and surface plasmon resonance). Despite the 'decent' affinity, we couldn't purify an homogeneous complex in size exclusion chromatography, even mixing the protein at concentrations up to 80-100 uM each. We explained this behavior by assuming that extremely high Kon/Koff values combine to give this 10 uM affinity, and the high Koff value would account for the dissociation going on during size exclusion chromatography. We have partial evidence for this from the SPR curves, although we haven't actually measured the Kon/Koff values. We eventually managed to solve the crystal structure of the complex by mixing the two proteins (we had to add an excess of one of them to get good diffraction data). Once solved the structure (which makes perfect biological sense and has been validated), we get mean B factors for one of the component (the larger) much lower than those of the other component (the smaller one, which we had in excess). We're talking about 48 Å^2 vs. 75 Å^2. I was wondering if anybody has had some similar cases, or has any hint on the possible relationship it might (or might not) exist between high a Koff value and high B factors (a relationship we are tempted to draw). Thanks in advance, best regards, ciao s -- Sebastiano Pasqualato, PhD IFOM-IEO Campus Dipartimento di Oncologia Sperimentale Istituto Europeo di Oncologia via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5094 fax +39 02 9437 5990
