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

native purification

before you start, buffers needed:

i015t8.5n500z : 15 mM imidazole, 10 mM tris, pH 8.5, 500 mM NaCl, 10 uM ZnSO4

i030t8.5n500z : 30 mM imidazole, 10 mM tris, pH 8.5, 500 mM NaCl, 10 uM ZnSO4

i500t8.5n500z : 500 mM imidazole, 10 mM tris, pH 8.5, 500 mM NaCl, 10 uM ZnSO4

1M DTT

1M benzamidine

(1) add 25 mL of i015t8.5n500z into a frozen cell pellet and thaw.

(2) sonicate in ice bath

(3) spin down cell pellet

(4) transfer supernatant into new falcon tube and add 3 mL of nickel beads

(5) rock the tube for at least 30 minutes in cold.

(6) spin down the beads and discard the supernatant

(7) wash the beads with i015t8.5n500z twice and with i030t8.5n500z twice

(8) in the final i030t8.5n500z wash,  pour the beads unto gravity filter column

(9) elute the protein with i500t8.5n500z

(10) add benzamidine, and add DTT, if the protein has cysteines