Le Vendredi 17 Mars 2017 16:07 CET, Nicholas Larsen <nicholas_lar...@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.]
