I recall a case of this a few years ago where it had to do with the relative concentration of the protein to the buffer/salt molecules (can't remember which anymore), which ended up being important in the binding that was observed. So it was entirely a concentration effect that caused the difference, where using the higher concentrations for ITC caused an increase in the observed Kd.
Scott On Fri, Mar 17, 2017 at 1:47 PM, DUMAS Philippe (VIE) < [email protected]> wrote: > > Le Vendredi 17 Mars 2017 16:07 CET, Nicholas Larsen <nicholas_larsen@ > H3BIOMEDICINE.COM> a écrit: > > Do you mean that the affinity from ITC is 100-fold weaker ? Which would > mean that the Kd values from ITC are 100-fold larger (in the range 0.1-1 > µM) ? > If this is the case, it makes me think to a problem that we have observed > by comparing Kd values obtained by ITC and by Mass Spec. The Kd values by > mass spec were very well determined and smaller (higher affinity) than > those obtained by ITC after a classical processing. It turns out that > processing the ITC data with two competing modes of binding revealed the > correct higher affinity binding mode observed by Mass Spec (80 %) mixed > with the lower-affinity binding mode (20 %). The a priori very strange > thing, which we finally explained, is that the initial processing of the > ITC data with only one binding mode had mixed wrongly the Kd for the > low-affinity binding mode, but the DeltaH for the high-affinity binding > mode. > > I suggest that you have a look to our paper: Wolff et al., J Am Soc Mass > Spectrom. 2017; 28(2): 347–357 (Open access). > I hope it will help. > Philippe Dumas > > > Dear colleagues, > > We have a target where people have measured Kd's for ligands using > > radioligand binding assays. Several publications report Kd's of single > > digit nanomolar and we are able to reproduce that data using this assay > > format. When we try to do the same measurement using ITC, we generate > > beautiful data, but the Kd's from ITC are at least 100-fold weaker. Does > > anyone have a suggestion how to reconcile this huge difference? > > > > SPR studies show the ligands have a very long residence time, so one > thing > > I wondered is if ITC can underestimate a Kd if the off-rate is on the > order > > of minutes-hours. Is this a reasonable explanation? > > > > Please, any other ideas are welcome. > > Best, > > Nick > > > > -- > > [This e-mail message may contain privileged, confidential and/or > > proprietary information of H3 Biomedicine. If you believe that it has > been > > sent to you in error, please contact the sender immediately and delete > the > > message including any attachments, without copying, using, or > distributing > > any of the information contained therein. This e-mail message should not > be > > interpreted to include a digital or electronic signature that can be used > > to authenticate an agreement, contract or other legal document, nor to > > reflect an intention to be bound to any legally-binding agreement or > > contract.] > > > > > -- Scott Horowitz, Ph.D. Postdoctoral Fellow University of Michigan Department of Molecular, Cellular, and Developmental Biology Bardwell lab 830 N. University Ave, Room 4007 Ann Arbor, MI 48109 phone: 734-647-6683 fax: 734-615-4226
