Well, mixing together has a much bigger pipetting error (to get, say, 500uM, you have to add 1000uM proteins A and B together, so with a pipetting error of ~1% even (and concentrated protein solutions seem to be tricky to pipette accurately), there would be an error of >10uM. Also, there are the errors associated with concentration determination, which are probably not trivial, especially with low EC. If, however, one of the components A is preloaded at low concentration (1-5uM, say), as I have recommended, the excess of that component with be exactly 1-5uM, assuming the complex was loaded with a slight excess of A. And this, as a percentage of the total 500uM, is much less than the various errors involved in mixing the two together.
So this would be the procedure: Mix A with B at a calculated stoichiometric ratio of 1.1:1.0 Preload the column with a low level of A just before injecting the complex Inject the complex Collect fractions, solve structure, publish in your favorite venue JPK On Fri, Nov 19, 2010 at 8:30 AM, <herman.schreu...@sanofi-aventis.com> wrote: > Dear Jacob, > The SEC is generally run to separate the complex from the unbound components. > If run the way your propose, the peak of unbound preinjected smaller > component coincides with the peak of the complex and the final stochiometry > is not better than by just mixing the components without SEC. > > Best, Herman > > -----Original Message----- > From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jacob > Keller > Sent: Friday, November 19, 2010 3:01 PM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] relationship between B factors and Koff > > A suggestion for purifying the complex: let's say there is a 5mL gap between > the complex and one of its (smaller)constituents A. You can pre-load the > column with, say, 5mL of A at 1uM, then inject the complex at 80-100uM, to be > injected right after the pre-load. This should provide approximately > equilibrium conditions, so that the complex should be basically 1:1 when it > comes out, even with a high Koff. (Alternatively, for true equilibrium > conditions, just equilibrate the entire column in A, then inject the > complex.) > > JPK > > ----- Original Message ----- > From: "Justin Hall" <hallj...@onid.orst.edu> > To: <CCP4BB@JISCMAIL.AC.UK> > Sent: Friday, November 19, 2010 7:32 AM > Subject: Re: [ccp4bb] relationship between B factors and Koff > > > Hi Sebastiano, > > I have had some experience with protein:protein complexes with KD ~ > 10-1 uM, kinetic characterization and trying to purify a complex of > these proteins using SEC. While I would say that if you have reliable > evidence from SPR that you have a fast on (high Kon), then you must > have a fast off (high Koff) because by definition KD = 10 E-6 = > Koff/Kon. However, I have observed several systems where you have a KD > ~ 10-1 uM, but the kinetics are not fast on/fast off. In my > experience, I have never seen anything in the crystal structures of > the weak affinity complexes I have solved that would coorelate > B-factors to Kon/Koff, and while it might be tempting for you to draw > this comparison in your structure, I would warn that this is too large > a leap without further (non-anecdotal) evidence. > > As a further note, during SEC purification of complexes, I have > observed that you generally have to have the complexes at at least 5 > to 10-fold higher initial concentration if you want to purify the > complex, which you are only pushing with your 80-100 uM high end > concentration. A colleague of mine once told me this is due to a 5 to > 10-fold dilution effect upon addition to the column, but I have never > verified this nor read any primary source that validated this so I > cannot supply a reference (others might be able to help here). Good > luck and cheers~ > > ~Justin > > > > Quoting Sebastiano Pasqualato <sebastiano.pasqual...@ifom-ieo-campus.it>: > >> 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 >> >> > > > ******************************************* > Jacob Pearson Keller > Northwestern University > Medical Scientist Training Program > Dallos Laboratory > F. Searle 1-240 > 2240 Campus Drive > Evanston IL 60208 > lab: 847.491.2438 > cel: 773.608.9185 > email: j-kell...@northwestern.edu > ******************************************* >
