1D screening: Difference between revisions
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== 1D <sup>1</sup>H NMR Screening == | == 1D <sup>1</sup>H NMR Screening == | ||
Initial screening of NMR samples can be done with 1D <sup>1</sup>H NMR on | Initial screening of NMR samples can be done with 1D <sup>1</sup>H NMR on labeled or unlabeled 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 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. | 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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The criteria for judging the NMR spectrum are: | The criteria for judging the NMR spectrum are: | ||
#signal to noise as compared to the DSS peak | #good 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 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 is good (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 | #peaks should not be too broad, allowing resolved peaks to be observed | ||
# uniform intensity of peaks in the amide regions is ideal. | #uniform intensity of peaks in the amide regions is ideal. | ||
<br> | <br> | ||
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. <ref>Rossi, P. et. al. (2009). "A microscale protein NMR sample screening pipeline." ''J. Biomol. NMR'', '''in press.</ref> <br> <br> Samples with promising 1D spectra should be <sup>15</sup>N labeled so that the [[Nhsqc screen|[15N-1H] HSQC]] can be recorded. <br> | |||
== References == | |||
<references /> | |||
Latest revision as of 19:39, 16 November 2009
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:
- good 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 is good (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.
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. [1]
Samples with promising 1D spectra should be 15N labeled so that the [15N-1H] HSQC can be recorded.
References
- ↑ Rossi, P. et. al. (2009). "A microscale protein NMR sample screening pipeline." J. Biomol. NMR, in press.