NESG Wiki - User contributions [en]

Sedimentation equilibrium

2009-11-19T19:34:27Z

Aayee:

We use gel filtration chromatography to determine the protein oligomerization state in solution. 

equipment: biologic duo flow; column: Superdex 75 Hiload 16/60

Protein concentration

2009-11-19T19:05:41Z

Aayee:

== Rutger's protein concentration measurements ==

Coming soon...

== University of Toronto protein concentration measurements. ==

since july of 2009, we use thermofisher nanodrop1000 to measure the protein absorbance at 280 nm. the molecular weight and theoretical extinction coefficient is calculated using expasy.

Protein purification

2009-11-13T17:30:53Z

Aayee:

== 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 <ref>Snyder, D, et.al. (2005). “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'', '''127:''' 16505-16511. [http://www.ncbi.nlm.nih.gov/pubmed/16305237 pmid = 16305237] </ref>:

 

*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 (Arrowsmith proteomics NMR lab) Buffers ==

standard screening NMR buffers are: a5.0n300zd : 10 mM sodium acetate, pH 5.0, 300 mM NaCl, 10 uM ZnSO4, 10 mM DTT, 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail, 5% D2O 

m6.5n450zd: 10 mM MOPS, pH 6.5, 450 mM NaCl, 10 uM ZnSO4, 10 mM DTT, 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail, 5% D2O

t7.7n500zd : 10 mM tris, pH 7.7, 500 mM NaCl, 10 uM ZnSO4, 10 mM DTT, 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail, 5% D2O 

final nmr buffer for 13c/15n labelled sample depends on the protein of interest. all nmr buffers always contain : 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail.

(1) if the protein has no cysteine in the sequence, do not bother to add ZnSO4 and DTT (Zn ion will just be a nuisance and deuterated DTT is expensive).

== Typical University of Toronto Protein Purification Protocol ==

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 

(11) exchange buffer by concentrating, diluting with new buffer, reconcentrating in a vivaspin concentrator.

 

if it is 13c/15n sample, add step

(9a) put sample in dialysis bag with protease to cut his-tag and dialyse  against  cleavage buffer

(9b) pass the sample through nickel beads again, then follow step (10) above.

== References ==

<references />

Protein purification

2009-11-13T16:48:15Z

Aayee:

== 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 <ref>Snyder, D, et.al. (2005). “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'', '''127:''' 16505-16511. [http://www.ncbi.nlm.nih.gov/pubmed/16305237 pmid = 16305237] </ref>:

 

*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 (Arrowsmith proteomics NMR lab) Buffers ==

standard screening NMR buffers are: a5.0n300zd : 10 mM sodium acetate, pH 5.0, 300 mM NaCl, 10 uM ZnSO4, 10 mM DTT, 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail, 5% D2O 

m6.5n450zd: 10 mM MOPS, pH 6.5, 450 mM NaCl, 10 uM ZnSO4, 10 mM DTT, 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail, 5% D2O

t7.7n500zd : 10 mM tris, pH 7.7, 500 mM NaCl, 10 uM ZnSO4, 10 mM DTT, 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail, 5% D2O

final nmr buffer for 13c/15n labelled sample depends on the protein of interest. all nmr buffers always contain : 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail. 

if the protein has no cysteine in the sequence, do not bother to add ZnSO4 and DTT (Zn ion will just be a nuisance and deuterated DTT is expensive).

== Typical University of Toronto Protein Purification Protocol ==

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 

(11) exchange buffer by concentrating, diluting with new buffer, reconcentrating in a vivaspin concentrator.

 

if it is 13c/15n sample, add step

(9a) put sample in dialysis bag with protease to cut his-tag and dialyse  against  cleavage buffer

(9b) pass the sample through nickel beads again, then follow step (10) above.

== References ==

<references />

Protein purification

2009-11-13T16:43:00Z

Aayee:

== 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 <ref>Snyder, D, et.al. (2005). “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'', '''127:''' 16505-16511. [http://www.ncbi.nlm.nih.gov/pubmed/16305237 pmid = 16305237] </ref>:

 

*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 (Arrowsmith proteomics NMR lab) Buffers ==

standard screening NMR buffers are: a5.0n300zd : 10 mM sodium acetate, pH 5.0, 300 mM NaCl, 10 uM ZnSO4, 10 mM DTT, 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail, 5% D2O 

m6.5n450zd: 10 mM MOPS, pH 6.5, 450 mM NaCl, 10 uM ZnSO4, 10 mM DTT, 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail, 5% D2O

t7.7n500zd : 10 mM tris, pH 7.7, 500 mM NaCl, 10 uM ZnSO4, 10 mM DTT, 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail, 5% D2O

final nmr buffer for 13c/15n labelled sample depends on the protein of interest. all nmr buffers always contain : 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail. 

if the protein has no cysteine in the sequence, do not bother to add ZnSO4 and DTT (Zn ion will just be a nuisance and deuterated DTT is expensive).

== Typical University of Toronto Protein Purification Protocol ==

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 it is 13c/15n sample, add step

(9a) put sample in dialysis bag with protease to cut his-tag cleavage and dialyse  against  cleavage buffer

(9b) pass the sample through nickel beads again, then follow step (10) above.

== References ==

<references />

Protein purification

2009-11-13T16:34:22Z

Aayee:

== 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 <ref>Snyder, D, et.al. (2005). “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'', '''127:''' 16505-16511. [http://www.ncbi.nlm.nih.gov/pubmed/16305237 pmid = 16305237] </ref>:

 

*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 (Arrowsmith proteomics NMR lab) Buffers ==

standard screening NMR buffers are: a5.0n300zd : 10 mM sodium acetate, pH 5.0, 300 mM NaCl, 10 uM ZnSO4, 10 mM DTT, 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail, 5% D2O 

m6.5n450zd: 10 mM MOPS, pH 6.5, 450 mM NaCl, 10 uM ZnSO4, 10 mM DTT, 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail, 5% D2O

t7.7n500zd : 10 mM tris, pH 7.7, 500 mM NaCl, 10 uM ZnSO4, 10 mM DTT, 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail, 5% D2O

final nmr buffer for 13c/15n labelled sample depends on the protein of interest. all nmr buffers always contain : 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail. 

if the protein has no cysteine in the sequence, do not bother to add ZnSO4 and DTT (Zn ion will just be a nuisance and deuterated DTT is expensive).

== Typical University of Toronto Protein Purification Protocol ==

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

== References ==

<references />

Protein purification

2009-11-13T15:41:09Z

Aayee:

== 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 <ref>Snyder, D, et.al. (2005). “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'', '''127:''' 16505-16511. [http://www.ncbi.nlm.nih.gov/pubmed/16305237 pmid = 16305237] </ref>:

 

*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 ==

standard screening NMR buffers are: a5.0n300zd : 10 mM sodium acetate, pH 5.0, 300 mM NaCl, 10 uM ZnSO4, 10 mM DTT, 0.01 % NaN3, 1 mM benzamidine 

'''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 ==

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

== References ==

<references />

Protein purification

2009-11-13T15:31:26Z

Aayee:

== 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 <ref>Snyder, D, et.al. (2005). “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'', '''127:''' 16505-16511. [http://www.ncbi.nlm.nih.gov/pubmed/16305237 pmid = 16305237] </ref>:

 

*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 ==

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

== References ==

<references />

Protein purification

2009-11-13T15:30:34Z

Aayee:

== 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 <ref>Snyder, D, et.al. (2005). “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'', '''127:''' 16505-16511. [http://www.ncbi.nlm.nih.gov/pubmed/16305237 pmid = 16305237] </ref>:

 

*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

== References ==

<references />

Protein purification

2009-11-13T15:29:07Z

Aayee:

== 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 <ref>Snyder, D, et.al. (2005). “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'', '''127:''' 16505-16511. [http://www.ncbi.nlm.nih.gov/pubmed/16305237 pmid = 16305237] </ref>:

 

*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

== References ==

<references />