Re: [ccp4bb] offtopic: effect of compound impurities on ITC?
Hi Francis I guess it depends on how much residual high-affinity binder you have in the mixture and what the difference in affinity is between Y and deriv-Y. Another issue is of course whether Y and derY compete for the same binding site and have the same stoichiometry. A well designed displacement ITC experiment and comparisons thereof with ITC data for your high-affinity binder should lead to some good answers. Knowing the ratio of Y vs deriv-Y in your starting compound solution will be an advantage. A very useful reference in thinking about and carrying out displacement ITC in our group has been the one by Velazquez-Campoy and Freire. This article was specifically written to address the application of displacement titrations in ITC. We have applied this approach to address several types of questions concerning interactions in the uM-pM range. Velazquez-Campoy A, Freire E. Isothermal titration calorimetry to determine association constants for high-affinity ligands Nat Protoc. 2006;1(1):186-91. Best regards Savvas Savvas Savvides Unit for Structural Biology @ L-ProBE Ghent University K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Ph. +32 (0)472 928 519 http://www.LProBE.ugent.be/xray.html On 24 Aug 2010, at 17:11, Francis E Reyes wrote: Hi All I'm curious the effect of small impurities in commercially synthesized compounds on ITC and its analysis. Say if compound Y is the high affinity binder, but you make a derivative that differs from a single functional group from Y (you used Y to make this new compound) and you never are able to completely get rid of Y. How does this affect the analysis of determining the derivative's affinity by ITC? References or personal experience is appreciated! F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
Re: [ccp4bb] offtopic: effect of compound impurities on ITC?
Hi Francis, I might save you some time by telling you up front you should just go back and purify your compound to remove the impurity, you dont even need to read the rest of this, just go. Along the lines of what Savvas was saying, with any equilibrium binding assay between two direct competitors (Y is the impurity and Z is your analyte), if you are working at concentrations above the KD then the resultant complexes (XY and XZ) will partition according to their relative association strengths (dG) and concentrations. So, if Y and Z have equivalent dG values, then the concentration of XY ([XY]) and [XZ] will be a function of [Y] and [Z], if [Y]=[Z] in this circumstance then [XY]=[XZ]. If dGy dGz or [Y] [Z], then you are in the clear. This is why going back to purify Z from Y is a good idea. Now,the great thing about ITC is of course that you can get dG, dH and -TdS in one experiment, but this is also going to bite you in the butt here since you will simultaneously be determining dG, dH and -TdS for both Y and Z, which leaves you will more unknowns that you have data to solve for unless you independently know [X], [Y], [Z] and dG, dH and -TdS for XY or XZ. In fact, the circumstance where you know [X], [Y], [Z] and dG, dH and -TdS for XY or XZ is what Savvas is describing with displacement assays, and unless I am misunderstanding your situation it sounds like you dont know these parameters. For that reason I would not qualify this as a displacement assay, but instead just as a poorly controlled experiment . Now, you might be able to do the experiment with pure Y binding to X to determine dG, dH and TdS, then perform the proposed experiment with impure Y and Z as a displacement binding, but this is going to still be a headache because your uncertainty will be greater, you will not have as accurate a measure of [Y] and [Z] as when they are pure, and since your your direct signal (dH) is going to be from the formation of both XY and XZ (dHtotal = dHy + dHz) S/N will be equal to or less than the experiment with pure Y or pure Z (my nanny used to say 'dont do good experiments with bad reagents, youll just waste time', she was very wise). Hope that helps, cheers~ ~Justin Quoting Savvas Savvides savvas.savvi...@ugent.be: Hi Francis I guess it depends on how much residual high-affinity binder you have in the mixture and what the difference in affinity is between Y and deriv-Y. Another issue is of course whether Y and derY compete for the same binding site and have the same stoichiometry. A well designed displacement ITC experiment and comparisons thereof with ITC data for your high-affinity binder should lead to some good answers. Knowing the ratio of Y vs deriv-Y in your starting compound solution will be an advantage. A very useful reference in thinking about and carrying out displacement ITC in our group has been the one by Velazquez-Campoy and Freire. This article was specifically written to address the application of displacement titrations in ITC. We have applied this approach to address several types of questions concerning interactions in the uM-pM range. Velazquez-Campoy A, Freire E. Isothermal titration calorimetry to determine association constants for high-affinity ligands Nat Protoc. 2006;1(1):186-91. Best regards Savvas Savvas Savvides Unit for Structural Biology @ L-ProBE Ghent University K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Ph. +32 (0)472 928 519 http://www.LProBE.ugent.be/xray.html On 24 Aug 2010, at 17:11, Francis E Reyes wrote: Hi All I'm curious the effect of small impurities in commercially synthesized compounds on ITC and its analysis. Say if compound Y is the high affinity binder, but you make a derivative that differs from a single functional group from Y (you used Y to make this new compound) and you never are able to completely get rid of Y. How does this affect the analysis of determining the derivative's affinity by ITC? References or personal experience is appreciated! F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
[ccp4bb] offtopic: effect of compound impurities on ITC?
Hi All I'm curious the effect of small impurities in commercially synthesized compounds on ITC and its analysis. Say if compound Y is the high affinity binder, but you make a derivative that differs from a single functional group from Y (you used Y to make this new compound) and you never are able to completely get rid of Y. How does this affect the analysis of determining the derivative's affinity by ITC? References or personal experience is appreciated! F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
Re: [ccp4bb] offtopic: effect of compound impurities on ITC?
Hi, If your are talking about proteins or protein subunits, this means that you are making polymers of nY, and Y becames the monomer. So in this case, I will not consider Y as an impurity. If I get you right, then size exclusion chromtography is good option to separate the monomers from the bigger polymers. Another way to do that, in my opinon, is that if you have a good estimate of the stoichometry between the monomers and polymers (and hence concentration of monomer in the cell), then run a refrence ITC with same monomer concetrate and this will automatically subtract the influence of Y component. I hope this give the slightest hint!! Ahmed. --- On Tue, 8/24/10, Francis E Reyes francis.re...@colorado.edu wrote: From: Francis E Reyes francis.re...@colorado.edu Subject: [ccp4bb] offtopic: effect of compound impurities on ITC? To: CCP4BB@JISCMAIL.AC.UK Date: Tuesday, August 24, 2010, 11:11 AM Hi All I'm curious the effect of small impurities in commercially synthesized compounds on ITC and its analysis. Say if compound Y is the high affinity binder, but you make a derivative that differs from a single functional group from Y (you used Y to make this new compound) and you never are able to completely get rid of Y. How does this affect the analysis of determining the derivative's affinity by ITC? References or personal experience is appreciated! F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
