1D screening: Difference between revisions
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Samples with promising 1D <sup>1</sup>H spectra should be <sup>15</sup>N labeled so that the [[Nhsqc screen|[<sup>15</sup>N-<sup>1</sup>H HSQC]] can be recorded. | Samples with promising 1D <sup>1</sup>H spectra should be <sup>15</sup>N labeled so that the [[Nhsqc screen|[<sup>15</sup>N-<sup>1</sup>H] HSQC]] can be recorded. | ||
Revision as of 19:03, 16 November 2009
1D 1H NMR Screening
Initial screening of NMR samples can be done with 1D 1H NMR on unlabelled 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 UB absorption and 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:
- signal to noise as compared to the DSS peak
- 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)
- chemical shifts dispersion in the amide region (will be much less if protein is predicted to be mostly helical vs. mosty beta strand.
Samples with promising 1D 1H spectra should be 15N labeled so that the [15N-1H] HSQC can be recorded.