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 NMR Buffers

The protein production facility at Rutgers University uses three typical NMR buffers for the initial protein screening.

They are :

• pH 4.5 NMR buffer:  20 mM NH₄OAc, 100 mM NaCl, 10 mM DTT, 5 mM CaCl2, 0.02% NaN₃, 5% D₂O
• pH 5.5 NMR buffer:  20 mM NH₄OAc, 100 mM NaCl, 10 mM DTT, 5 mM CaCl2, 0.02% NaN₃, 5% D₂O
• pH 6.5 NMR buffer:  20 mM MES, 100 mM NaCl, 5 mM CaCl₂, 10 mM 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...