Protein purification

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 :

• 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.” 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