The application of the transferred NOE (Tr-NOE) effect was first demonstrated by Bothner-By [282]. The Tr-NOE is the nuclear Overhauser effect between ligand spins, which are in chemical exchange between the bound and unbound form with the protein or receptor. Ligands, which are a mixture of target molecules, are small in size (below 500–1000 Da). Since they are usually low molecular weight molecules, they exhibit much shorter correlation times when compared to the receptor and have slow NOE build-ups with no spin diffusion. This is the reason they show small positive NOEs in the free form. When binding to a protein receptor, the situation changes, where the ligand acquires large correlation times in the bound state with rapid NOE build-up. Then they show spin diffusion and a strong negative NOE, which is termed the transferred NOE. Signals arising from the protein are usually not observed for large proteins as they are generally kept low in concentration, with ligands in a high excess concentration. In addition, most of the time protein signals are suppressed by their very short T2 period. It is worthwhile to mention that ligands that are in fast exchange between the bound and the free form (dissociation constants ranging from μM to mM) get enough bound time to transfer the negative NOE from the protein complex to the population of the free molecules, yet usually retain the chemical shift of the free molecule along with the relaxation characteristics. In order to observe tr-NOEs, the following condition have to be fulfilled:(3) |Nb∂b|≫|Nf∂f| where N and ∂ represent the number of molecules and the cross-relaxation rate, respectively. The subscript b and f represent the bound and free form, respectively. Therefore, to observe the tr-NOEs, a high excess concentration of ligands over protein is maintained. On the other hand, if the ligand concentration is kept too high, the excess free ligand in solution will exhibit positive NOE, which can result in a significant reduction of the tr-NOESY enhancements due to negative NOE developed by the very small concentration of bound ligand. Hence, the preparation of the sample becomes tricky and an optimum ratio between 10–30 to 1 is maintained while considering the dissociation constant values. The binding of a ligand to a receptor protein can easily be identified by observing the sign and size of the NOEs. There are some distinct experimental features for the discrimination between tr-NOEs from the bound state and NOEs of the ligand in free states like the build-up rate. For tr-NOEs, this is in the range of 50 to 100 ms, whereas for small ligands it is much longer. There have been various instances of experimental implementations to quickly determine the binding activity of ligand libraries. One example was to find the ligand molecule among a library of 10 similar structure polysaccharides that is bioactive in binding with recombinant E-selectin [283]. This is a protein present in an IgG chimera with a molecular weight of about 220 kDa. In this case, two 2D NOESY spectra were recorded. The NOESY spectra for the ligand library was measured at several temperatures and it was found that most of the 10 compounds exhibited the weak positive NOEs at 310 K, which was then chosen to differentiate between trNOEs showing large negative values. The trNOESY spectra of the ligand library in the presence of protein was recorded at different ratios, such as 5:1, 8:1, 12:1, 15:1, and 20:1, at 310 K. In all the ratios, trNOEs were observed; however, the ratio of 15:1 represented the best-case scenario.