Protein purification: Difference between revisions
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Samples for NMR
Typical Rutgers University NMR Buffers
Typical Rutgers University Protein Purification Protocol
Typical University of Toronto NMR Buffers
Typical University of Toronto Protein Purification Protocol
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They are <ref>Snyder, D, et.al. “Comparisons of NMR spectral quality and success in crystallization demonstrate that NMR and X-ray crystallography are complementary methods for small protein structure determination.” JACS, | They are <ref>Snyder, D, et.al. (2005). “Comparisons of NMR spectral quality and success in crystallization demonstrate that NMR and X-ray crystallography are complementary methods for small protein structure determination.” ''JACS'', '''127:''' 16505-16511.pmid = </ref>: | ||
*pH 4.5 NMR buffer:<span style=""> </span>20mM | <ref>{{cite journal | author = R.D. Johnson, L. Johnson, Y. Itoh, M. Kodama, H. Otani, and K. Kohmoto | year = 2000 | journal = Molecular Plant-Microbe Interactions | volume = 13 | pages = 742-753 | url = http://apsjournals.apsnet.org/doi/abs/10.1094/MPMI.2000.13.7.742?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%3dncbi.nlm.nih.gov | pmid = 10875335 | doi = 10.1094/MPMI.2000.13.7.742 | title = Cloning and Characterization of a Cyclic Peptide Synthetase Gene from Alternaria alternata Apple Pathotype Whose Product Is Involved in AM-Toxin Synthesis and Pathogenicity}}</ref> | ||
*pH 5.5 NMR buffer:<span style=""> </span>20mM | |||
*pH 6.5 NMR buffer:<span style=""> </span>20mM MES, 100mM NaCl, | *pH 4.5 NMR buffer:<span style=""> </span>20mM NH<sub>4</sub>OAc, 100mM NaCl, 10mM DTT, 5mM CaCl2, 0.02% NaN<sub>3</sub>, 5% D<sub>2</sub>O | ||
*pH 5.5 NMR buffer:<span style=""> </span>20mM NH<sub>4</sub>OAc, 100mM NaCl, 10mM DTT, 5mM CaCl2, 0.02% NaN<sub>3</sub>, 5% D<sub>2</sub>O | |||
*pH 6.5 NMR buffer:<span style=""> </span>20mM MES, 100mM NaCl, 5 mM CaCl<sub>2</sub>, 10mM DTT, 0.02% NaN<sub>3</sub>, 5% D<sub>2</sub>O | |||
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Revision as of 20:26, 12 November 2009
Samples for NMR
For each protein, we usually make the following samples:
- 100% 15N, 100% 13C uniform labeled sample, for resonance assignment and NOE interpretion.
- 100% 15N, 5% 13C labeled sample, for stereospecific assignment of VAL and LEU isopropyl moieties.
For RDC measurement:
- A secondary 100% 15N, 5% 13C labeled sample, for RDC measurement.
For each protein that exist as dimer in solution an extra sample may be required in addition to the samples above:
- 1:1 unlabeled and 100% 15N, 100% 13C uniformed labeled mixed sample, for intermolecular NOE interpretation
Typical Rutgers University NMR Buffers
The protein production facility at Rutgers University uses three typical NMR buffers for the initial protein screening.
They are [1]:
- pH 4.5 NMR buffer: 20mM NH4OAc, 100mM NaCl, 10mM DTT, 5mM CaCl2, 0.02% NaN3, 5% D2O
- pH 5.5 NMR buffer: 20mM NH4OAc, 100mM NaCl, 10mM DTT, 5mM CaCl2, 0.02% NaN3, 5% D2O
- pH 6.5 NMR buffer: 20mM MES, 100mM NaCl, 5 mM CaCl2, 10mM DTT, 0.02% NaN3, 5% D2O
Typical Rutgers University Protein Purification Protocol
Coming soon...
Typical University of Toronto NMR Buffers
Some typical NMR buffers are:
t7.3n250zd buffer: 10 mM tris, 250 mM NaCl, ~10 uM Zn++, 10 mM DTT, 0.01% NaN3,1 mM benzamidine, 1x inhibitor cocktail, 5% D2O, pH 7.3
m6.5n450zd buffer: 10 mM MOPS, 450 mM NaCl , ~10 uM Zn++, 10 mM DTT, 0.01% NaN3,1 mM benzamidine, 1x inhibitor cocktail, 5% D2O, pH 6.5
Typical University of Toronto Protein Purification Protocol
coming soon...
References
- ↑ Snyder, D, et.al. (2005). “Comparisons of NMR spectral quality and success in crystallization demonstrate that NMR and X-ray crystallography are complementary methods for small protein structure determination.” JACS, 127: 16505-16511.pmid =
- ↑ Template:Cite journal