In addition to using TNP-ATP to determine whether a protein binds ATP, its binding affinity and dissociation constants, and number of binding sites, TNP-ATP can also be used in ligand binding studies. To do this, titrations of the protein are added to TNP-ATP. Then, ligand is added to displace the bound analog. This is measured by decreases in fluorescence. One can also do this by titrating protein with TNP-ATP in the presence and absence of varying concentrations of the ligand of interest. Using either experiment will allow the binding affinity of the ligand to protein to be measured.
TNP-ATP is also valuable fluorescence acceptor. This is because, as with any good acceptor, TNP-ATP absorbs over a wide wavelength range that corresponds to the range of emission of common FRET donors. Thus, TNP-ATP can be used to look at the conformational changes that proteins undergo.  For example, Na+/K+ ATPase, the distance between the active site and Cys457 was shown to change from 25 Angstroms to 28 Angstroms in changing from the Na+ conformation to the K+ conformation.
In addition to fluorescent spectroscopy, TNP-ATP is very useful in fluorescent microscopy. This is because it greatly increases the sensitivity of the observations when bound to proteins—the enhanced fluorescence greatly reduces the problem of background fluorescence. This is especially true under epifluorescent illumation (illumination and light are both on the same side of the specimen).
TNP-ATP has also been used in X-ray crystallography because it can be used to determine binding constants of crystallized substrates. This technique also demonstrates the structure of proteins in the presence or absence of TNP-ATP, which may or may not correspond to the structure of proteins when they bind ATP.