Protein purification: Difference between revisions
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== Typical Rutgers University | == Typical Rutgers University NMR Buffers<br> == | ||
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.” | 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.” <cite>JACS</cite>, v.127 issue 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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References | References | ||
<br> <references /> | <br> <references /> <br> 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. | ||
<br> | |||
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 23:14, 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 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, 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.” <cite>JACS</cite>, v.127 issue 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.
Typical University of Toronto Protocol
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