I should add that this procedure is really only advantageous for high Koff complexes. If the complex does not dissociate appreciably in the time required for SEC, I agree that there is no great benefit for doing it my way. I have been working recently, however, on a high Koff complex, so have been thinking about how to get exactly the right ratio (other suggestions welcome!)
Jacob On Fri, Nov 19, 2010 at 8:45 AM, Jacob Keller <j-kell...@fsm.northwestern.edu> wrote: > 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 >> ******************************************* >> >
