If the peptide is really small compared to the protein, I would just add excess 
peptide and not worry about the stochiometry.


-----Original Message-----
From: Jacob Keller [mailto:j-kell...@fsm.northwestern.edu] 
Sent: Friday, November 19, 2010 5:03 PM
To: Schreuder, Herman R&D/DE
Cc: CCP4BB@jiscmail.ac.uk
Subject: Re: [ccp4bb] relationship between B factors and Koff

Well, if one of the components is a small peptide and the solutions are dilute, 
it is hard to concentrate the complex, as the free peptide will go through the 
concentrator! It's a tricky problem.


On Fri, Nov 19, 2010 at 9:38 AM,  <herman.schreu...@sanofi-aventis.com> wrote:
> 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,
> Herman
> -----Original Message-----
> From: Jacob Keller [mailto:j-kell...@fsm.northwestern.edu]
> Sent: Friday, November 19, 2010 3:57 PM
> To: Schreuder, Herman R&D/DE
> Cc: CCP4BB@jiscmail.ac.uk
> 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!)
> 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
>>> 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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