It depends on why you don't get a sigmoidal curve. If your protein is saturated after 1 or 2 injections, you need to either lower your injection volume or lower the concentration because you're not catching the inflection point. If you don't get a sigmoidal curve because the interaction is just too weak, you need to increase the concentration.

You should start in the neighbourhood of 10-30uM for your sample and 100-300uM for your ligand and then lower or raise the concentration according to your first experiment.

In your case, you should see if this heat of injection is simply due to the dilution caused by your injection, which can mean that the interaction is so weak that you need to go much higher in concentration in order to see an interaction.

Also, did you blank your experiment by injecting only buffer in a separate experiment so you can subtract the blank from your experiment? That can give you further insights.

The selection of ligand and receptor concentrations for an ITC experiment can make use of the parameter called C-window. It is described in this document, on page 3:

http://www.tainstruments.com/pdf/literature/MCAPN-2016-1.pdf

If you have a rough idea about stoichiometry and Kd, you can also use the prediction tool in the Malvern package. But I agree with Michael Adams that, usually, a good starting point is 20 uM cell and 200 uM syringe.

In addition to ensuring that the reactant concentrations are appropriate as suggested by others, a few other questions come to mind:

1. Did you dialyse the protein sample and dissolve the ligands in the dialysate? Are the buffer compositions of protein and ligand identical? Are the dilution heats so high that obscure the binding profile?

You should run controls as suggested by Michael, using the ligand in the syringe and dialysate (buffer) in the cell, and if possible also buffer in the syringe and protein in the cell at the same concentrations as those used in the experimental run.

2. Did you test several temperatures to ensure you are not at a temperature where the binding enthalpy is zero? This occurs often in the 20-30 ºC range and generally happens for systems that exhibit negative heat capacity changes and therefore are endothermic at low temperatures and become exothermic at higher temperatures.

I very much agree with Conceicao Minetti's remarks. Also it is good to test the range of temperatures because many ligand binding interactions have evolved which have no effect at the usual biological temperature of the organism. For anything from a human or from E. coli that is native to the human digestive tract, you will find many hidden interactions that have free energy of zero at 37 C, due to enthalpy-entropy compensation. They often evolve with the enthalpy being zero just below that temperature (the 20 to 30 C range). Others evolve with the enthalpy being zero above that range (45 to 55 C, and these are entropy-dominated binding events). Since you tend to lose binding itself at 37 C (i.e. free energy goes to zero), you can see more events if you are colder. If you go hotter, proteins that don't come from thermophilic bacteria often unfold.

Chakrabarti, S., Bryant, S. H., & Panchenko, A. R. (2007). Functional specificity lies within the properties and evolutionary changes of amino acids. Journal of molecular biology, 373(3), 801-810.

Breslauer, K. J., Remeta, D. P., Chou, W. Y., Ferrante, R., Curry, J., Zaunczkowski, D., ... & Marky, L. A. (1987). Enthalpy-entropy compensations in drug-DNA binding studies. Proceedings of the National Academy of Sciences, 84(24), 8922-8926

Waldron, T. T., & Murphy, K. P. (2003). Stabilization of proteins by ligand binding: application to drug screening and determination of unfolding energetics. Biochemistry, 42(17), 5058-5064.

From my experience, and in agreement with some of the answers, I would say that:

- You must perform control experiments injecting titrant into buffer to assess the heat effect produced by injection and ligand dilution.

- You must perform experiments under different conditions (e.g., temperature, pH...) to check if enthalpy is close to zero under the conditions you first selected.

- You must evaluate if the injections peaks, being similar, are too large (so they do not correspond to just unspecific injectioin background heat).

- You must guarantee an almost perfect match between titrant and titrand solutions, to avoid large unspecific heat effects.

- You should not expect to get always a sigmoidal binding isotherm. Sigmoidicity (or presence of inflection point) depends on the binding affinity and titrand concentration. If c = Pt x Ka (Pt, titrand concentration; Ka, association constant) is smaller than 1, there will be no inflection point. Of course, a sigmoidal isotherm is convenient (for many reasons), and, for a given affinity, increasing concentrations would result in a larger c-parameter and higher sigmoidicity; however, that might be impractical (too high concentrations). Small c-parameter does not mean the titration is useless, but it means that you must be even more careful in the data analysis than usual.

- You must periodically perform experiments with well-known reactants in order to test the equipment.

I am sure other recommendations could be added here.

For a standard 1:1 interaction, the titrand solution should be around 10-20 microM, and the titrant solution should be 100-200 microM (more or less, 10-fold concentration). Depending on the binding enthalpy, these concentrations can be scaled up or down. Depending on the binding affinity the titrant concentration should be ajusted: low affinity would require 20- or even 50-fold higher titrant concentration compared to titrand. If the stoichiometry is different than 1:1, the concentrations should be appropriately modified.