If the peptide is really small compared to the protein, I would just add excess peptide and not worry about the stochiometry.
Best, Herman -----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. JPK 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 >>> To: CCP4BB@JISCMAIL.AC.UK >>> 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> >>> To: <CCP4BB@JISCMAIL.AC.UK> >>> 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 >>> ******************************************* >>> >> >