1D screening: Difference between revisions

From NESG Wiki
Jump to navigation Jump to search
m (Created page with '== 1D <sup>1</sup>H NMR Screening == 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<su…')
 
mNo edit summary
 
(6 intermediate revisions by the same user not shown)
Line 1: Line 1:
== 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&nbsp;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&nbsp;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 UB absorption and 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.  


<br>  
<br>  
Line 9: Line 9:
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
#uniform intensity of peaks in the amide regions is ideal.


<br>  
<br>  


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.
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:

  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. [1]

Samples with promising 1D spectra should be 15N labeled so that the [15N-1H] HSQC can be recorded.

References

  1. Rossi, P. et. al. (2009). "A microscale protein NMR sample screening pipeline." J. Biomol. NMR, in press.