Place the protein sample in the superconducting magnet on the Nuclear Magnetic Resonance Spectrometer (NMR) by inserting it into the NMR probe. Then place the NMR probe into the bore of the superconducting magnet.
Using the Nuclear Magnetic Resonance computer workstation, tune the Nuclear Magnetic Resonance Spectrometer (NMR) to your sample for a two-dimensional experiment. Use “mtune” to adjust and give the best dip in your results.
Lock and shim the spectrometer to provide the best resolution and line shape in your sample.
Determine the 1H and 13C pw for the sample and assign sensitivity 90 for two-dimensional experiments. Do this by loading in the 1D and 1H proton experiment and tune and shim the sample to d1=10. Type in pw90? into the computer at the computer workstation to get the default value. For all NMR experiments, more concentrated protein samples require fewer scans.
Insert an additional indirect evolution time and a second mixing to the two-dimensional experiment to create a three-dimensional experiment. Do this after the first mixing and before the final data acquisition. The timeline for the three-dimensional experiment should be as follows: preparation, evolution, mixing time, evolution, mixing time and detection.
Repeat the steps for each protein sample you wish to test, making sure to add in the additional indirect evolution time and second mixing each time to create the three-dimensional experiment.