I don't think there is any binding. Your experiment at this time is not working, the eso and endothermic point are just bubbles in the syringe. You should repeat the experiment with different concentrations.

Thank for your answer Domenico.

But I don't think is bubble problem because in other experiments we've already had bubbles related problems. The bubbles resulted in different peak profile with lower heat exchange. We also degassed the cell and syringe buffer before the experiment to prevent this kind of problem.

Amanda - I agree with Dominico that there is likely no binding.   It would help me if I could see the actual data file from the MicroCal PEAQ-ITC, and can troubleshoot more.  You can email me directly at [email protected] 

Thank you, Verna Frasca.

I sent you an email. Hopefully, you can help me.

First: I have seen erratic titration profiles when injecting air into liquid. Are you using an automated calorimeter? In that case, check the system.

Second: Do you need gelatin 1%? Do you need DMF 0.5%? Do you need sodium azide 15 mM?

Third: Either you made a mistake writing or the experimental setting is not appropriate. You have 5.2 uM antibody in the calorimetric cell and 5 uM ligand in the syringe. Even if the binding affinity is high, this is not going to work because at the end of the sequence of injections the antibody will far from being saturated by ligand. According to your experimental parameters, at the end of the 39 injections there will be 4.28 uM antibody in the cell and 0.89 uM ligand in the cell. In case the affinity is very high, you will still have 20% of antibody in complex with ligand. Because you said the ligand is weak, then the fraction of antibody bound to ligand after the 39 injections will be much lower tan 20%. In the best of the situations you would just observe a sequence of more or less equal peaks.

Hi

1st i would like to say : try with different conc. u used almost same conc in both cell and syringe.generally the syringe conc should atleast 10-20 times more than cell conc. so adjust ur conc a/c this.

2nd thing your buffer.try with another buffer like phosphate buffer (it has low ionization value) may improve ur result. 1% gelatin, 15mM sodium azide, and 0,5% DMF (v/v) in ur buffer may be the reason for not getting result.

Adrian and Samima, thank for your opinions.

We reconstituted the lyophilized antibody following the seller protocol, then we separated into aliquots before freezing. The cell solution was prepared from the frozen aliquots, pooling and completing the volume with the same reconstitution buffer. The gelatin helps the antibody stabilization, the azide prevents microorganism activity and the DMF for ligand solubilization. In any case, we don't think the problem is with the buffer because the dilution heat test was fine.

We first performed an antigen-antibody titration with 50 uM antigen and 5 uM antibody. And the saturation was achieved in 2 injections. Then a lower concentration of antigen was tested (5 uM) and the same happened. And this is why we tested at those concentrations for the weak ligand too. In any case, we don't know why these positive and negative peaks keep showing.

And we don't think the problem is the equipment since the water-water and other protein-substrate titrations are fine.

We will repeat the experiment, probably with higher concentration. And we are studying methods to change the buffer without losing significant amounts of antibody.

Hi, did you solve your problem with endo and exo peaks at the same time? I also had this problem, I did itc with 70% DMSO as solvent, drug 0.4mM and cyclodextrin 0.05mM (in cell). it always have endo and exo peaks, and the DP had no rule. Iit’s our troubl. Thanks