1D screening: Difference between revisions
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Initial screening of NMR samples can be done with 1D <sup>1</sup>H NMR on unlabelled protein samples in aqueous buffer with 5-10% D<sub>2</sub>O. | Initial screening of NMR samples can be done with 1D <sup>1</sup>H NMR on unlabelled protein samples in aqueous buffer with 5-10% D<sub>2</sub>O. | ||
It is helpful to have an estimation of the protein concentration obtained from a method such as | 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. | ||
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#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 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. | #chemical shifts dispersion in the amide region (will be much less if protein is predicted to be mostly helical vs. mosty beta strand. | ||
# peaks should not be too broad, allowing resolved peaks to be observed | |||
# uniform intensity of peaks in the amide regions is ideal. | |||
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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 19:26, 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 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:
- 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.
- peaks should not be too broad, allowing resolved peaks to be observed
- uniform intensity of peaks in the amide regions is ideal.
Samples with promising 1D 1H spectra should be 15N labeled so that the [15N-1H] HSQC can be recorded.