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


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
> 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.)
> ----- Original Message -----
> From: "Justin Hall" <hallj...@onid.orst.edu>
> 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
> *******************************************

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