The 3D Sequential-HNCACB experiment is specifically designed to correlate the ¹H and ¹⁵N amide resonances with those of the inter-residue ¹³CA and ¹³CB resonances by means of the ¹J(NH), ²J(N,CA) and optional ¹J(CA,CB) coupling constants.

Implementation on AVANCE spectrometers equipped with a third channel. Improved versions using pulsed field gradients (PFGs) are also available and, therefore, in such cases gradient technology is required.

The experiment is applied on ¹⁵N,¹³C-labeled proteins. Because the amide (NH) protons are involved, the HNCO experiment must be recorded in H₂O.

The 3D intra-HNCACB experiment can be recorded in automation mode.

See list of 3D triple-resonance NMR experiments for doubly-labeled proteins.