The amount of small component one looses from the complex depends on the Koff 
and the local concentrations during the SEC run, so I doubt if one could 
estimate those losses with an error less than the 1% pipetting error you quote. 
What I would do is to pipet dilute solutions, and concentrate afterwards. If 
there are conditions which give bad crystals, one could also harvest these 
crystals, redissolve them and set up crystallizations with those.

My two cents,

-----Original Message-----
From: Jacob Keller [] 
Sent: Friday, November 19, 2010 3:57 PM
To: Schreuder, Herman R&D/DE
Subject: Re: [ccp4bb] relationship between B factors and Koff

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 <> 
> 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,  <> 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 [] 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" <>
>> 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 <>:
>>> 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:
>> *******************************************

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