Protein purification: Difference between revisions
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Samples for NMR
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The protein production facility at Rutgers University uses three typical NMR buffers for the initial protein screening. | The protein production facility at Rutgers University uses three typical NMR buffers for the initial protein screening. | ||
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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.” &amp;lt;cite&amp;gt;JACS&amp;lt;/cite&amp;gt;, v.&amp;amp;nbsp;127 issue&amp;amp;nbsp;47, 2005, p. 16505-16511.</ref>: | |||
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.” &lt;cite&gt;JACS&lt;/cite&gt;, v.&amp;nbsp;127 issue&amp;nbsp;47, 2005, p. 16505-16511.</ref>: | |||
*pH 4.5 NMR buffer:<span style=""> </span>20mM NH4OAc, 100mM NaCl, 10mM DTT, 5mM CaCl2, 0.02%NaN3, 5% D2O | *pH 4.5 NMR buffer:<span style=""> </span>20mM NH4OAc, 100mM NaCl, 10mM DTT, 5mM CaCl2, 0.02%NaN3, 5% D2O | ||
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*pH 6.5 NMR buffer:<span style=""> </span>20mM MES, 100mM NaCl, 5mM CaCl2, 10mM DTT, 0.02%, NaN3, 5% D2O | *pH 6.5 NMR buffer:<span style=""> </span>20mM MES, 100mM NaCl, 5mM CaCl2, 10mM DTT, 0.02%, NaN3, 5% D2O | ||
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References | References | ||
<br> <references /> 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.” <cite>JACS</cite>, v. | <br> <references /> 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.” <cite>JACS</cite>, v.127 issue 47, 2005, p. 16505-16511. | ||
== Typical University of Toronto Protocol == | == Typical University of Toronto Protocol == | ||
Revision as of 22:53, 11 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 Protocol
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, 5mM CaCl2, 10mM DTT, 0.02%, NaN3, 5% D2O
References
- ↑ 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.” &lt;cite&gt;JACS&lt;/cite&gt;, v.&amp;nbsp;127 issue&amp;nbsp;47, 2005, p. 16505-16511.
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, v.127 issue 47, 2005, p. 16505-16511.