Therefore, other approaches to rule out the effects of different buffer media are needed. Protonation effects have been demonstrated in numerous studies using ITC experiments, but this might only be suitable for ligands with an affinity K d <1mM and/or a high enthalpic contribution. However, for low affinity fragments, high resolution crystallography combined with neutron diffraction structure could be used. In addition, orthogonal methods for the binding detection could be used as well. We envision such experiments for our future studies on PKA-fragment complexes.