1D screening

1D ¹H NMR Screening

Initial screening of NMR samples can be done with 1D ¹H NMR on labeled or unlabeled protein samples in aqueous buffer with 5-10% D₂O.

It is helpful to have an estimation of the protein concentration obtained from a method such as UV absorption and also to add 50 uM DSS as an internal standard for both referencing (to 0 ppm) and for estimating the protein concentration.

The criteria for judging the NMR spectrum are:

1. good signal to noise as compared to the DSS peak
2. chemical shifts dispersion upfield of 0 ppm is good, since it indicates methyl protons that are in a folded core and nearby to methyl groups (not essential if the protein does not have aromatic residues)
3. chemical shifts dispersion in the amide region is good (will be much less if protein is predicted to be mostly helical vs. mosty beta strand.
4. peaks should not be too broad, allowing resolved peaks to be observed
5. uniform intensity of peaks in the amide regions is ideal.

At Rutger's University, samples are screened at 20 degrees using 1.7-mm NMR tubes that are loaded into a Bruker B-ACS 60 samples handler and run on a Bruker TCI 1.7 MicroCryoprobe.
Samples with promising 1D <sup>1</sup>H spectra should be <sup>15</sup>N labeled so that the [<sup>15</sup>N-<sup>1</sup>H] HSQC can be recorded.