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 
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


----- 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~


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

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