Two major problems are present in NMR of larger proteins: Overlapping resonances and increased linewidths. For this reason, isotopic labeling and 3D NMR experiments have been succesfully introduced to overcome such difficulties. Basically, the incorporation of ¹⁵N-labeling in proteins affords the possibility to expand the suite of NMR experiment to be applied. In addition to the conventional experiments described for unlabeled proteins, some new NMR experiments are usually applied taking advantage from the improved sensitivity for the ¹⁵N nucleus:

• Heteronuclear 2D ¹H-¹⁵N correlation experiments using proton detection as, for instance, HSQC, HMQC and TROSY experiments.
• Protein dynamics can be studied from ¹⁵N relaxation rates and from ¹H{¹⁵N} NOE data extracted from closely related HSQC and TROSY pulse trains.
• 2D and 3D NMR experiments such as, for instance, ¹H-¹⁵N TOCSY-HSQC (intraresidue assignments) or ¹H-¹⁵N NOESY-HSQC experiments (sequential NOE connectivities).
• 3D HNHA experiment or other methods to measure J_HN,HA coupling constants.
• 3D HNHB experiment to measure J_HA,HB coupling constants.

With the use of cryoprobes with high sensitivity, it is also feasible to acquire triple-resonance 3D experiments in proteins with 13C at natural abundance, as described for 15N,13C-doubly labeled proteins. For instance, when HNCA experiments are run on a natural abundance 13C sample, the magnetization of an amide NH proton is transferred to either inter or intra CA spins, but not both. In addition, there are not 13C-13C couplings and additional spin relaxation pathways. Thus, better sensitivity is obtained as predicted theoretically.