Since the K d of benzoic acid is not known, we have to probe different scenarios for different ranges of affinities. Assuming a K d of 1 mM corresponds to a saturation (D sat ) of about 80%, while a lower affinity of 10 mM saturates only 30% of the protein. As a rule of thumb, to achieve sufficient saturation of PKA or any other protein to be tested with the fragment during titration, the final concentration of the fragment added to the sample cuvette must be greater than its estimated K d value. In the case of benzoic acid, the concentration used would be correct for a hypothetical dissociation constant of 1 mM, whereas for a K d of 10 mM the fragment concentration should be higher. However, at even higher fragment concentrations, more non-specific binding may occur. This could be an explanation why no binding is observed in any of our ITC experiments.