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!)


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
> 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.)
>> JPK
>> ----- 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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