I want to investigate the interaction between two proteins by ITC. I choose two proteins that I'm sure don't interact with each other as a control. but ITC results show there is some interaction in control !how is it possible!?
Hi @Parastou Rahimizadeh
Such non-saturating curves (as in case of your control) could be observed due to slight buffer differences or sometimes, these heat changes also originate from self aggregation of either one of the two test proteins.
Yeshveer Singh Thanks for your reply
yeah, I think one of my interactants (protein in the syringe) form homodimer at high concentration. I should repeat ITC by each of the interactants separately to be sure. If you have any suggestions to get a better curve, I will be thankful.
@Parastou Rahimizadeh
You can keep the protein which forms homodimers inside cell instead of syringe. As you mentioned at lower concentration it is monomeric, so this reversal may work.
You can also add some component in buffer which keeps your protein in monomer form.
Otherwise as an alternative approach, you can use SPR.
I hope it will help.
Dear Yeshveer Singh
I did ITC again with a lower concentration of interacts and I didn't see any interaction in my control^^. but the interesting matter is that I saw differences Kd in my sample analysis. I thought due to the concentration independence of the Kd parameter, I won't see changes in Kd in the repetition of my sample analysis (i mean i repeat sample analysis also with lower concentration, but Kd changed!). do you have any idea?!
In the first "sample.pdf" the saturation occurred very soon, the binding isotherm was ill-defined, and the binding affinity could be incorrectly determined.
IN the second "sample.pdf" the binding isotherm is much better, and the binding affinity is more reliable.
In any case, the Kd is concentration independent (contrary to EC50 or IC50) if there are no oligomer-monomer equilibria coupled to the binding. If oligomer-monomer equilibrium is present for any of the recant molecules, and if there is preferential binding to oligomer or monomer, the (apparent) Kd will be concentration dependent, because that apparent Kd will implicitly contain the dependency on that additional equilibrium. If a refined model considering that self-association interaction explicitly in the apparent Kd is applied, then a concentration-independent Kd could be determined.
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