== Samples for NMR ==

For each protein, we usually make the following samples:

*[''U''-15N, 13C]-labeled sample, for resonance assignment and NOE interpretation.
*

A U-15N and fractional 5%-13C-labeled sample for stereospecific assignment of Val and Leu isopropyl methyl groups based on the method of Neri et. al. (Biochemistry 28, 7510-7516,1989) that we call the NC5 sample.  This sample is prepared using 5% ''U''[1H,13C]-D-glucose and 95% unlabelled glucose in the ''E. coli'' minimal  growth media.

 The NC5 sample is not [''U''-5%-13C,''U''-15N] since it is not uniformly 5% -13C labeled.  The incorporation of 13C in the methyl groups is biosynthetically directed by the ''E. coli''. 

This sample can be called: 

           ''U''-15N, 5%
biosynthetically directed 13C (NC5) sample or

            ''U''-15N, fractional 5%-13C-labeled (NC5) sample  

and give the Neri reference.

 For RDC measurement:

*A secondary NC5 sample for RDC measurement. 

(Alternatively, for the H-N RDC measurement, a [U-15N]-labeled sample can be used. Rutger's uses a second NC5 sample, since this is all ready being prepared for the stereospecific assignments, and a 13C CHSQC can be compared with the [U-15N, 13C]-labeled sample, to check if the samples are the same.)

 For each protein that is a dimer in solution an extra sample may be required in addition to the samples above:

*1:1 mixture of natural abundance and [U-15N, 13C]-labeled samples, for intermolecular NOE interpretation.

 

== Typical Rutgers University NMR Buffers ==

The protein production facility at Rutgers University uses these three NMR buffers for the initial protein screening: <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 ==

''E.coli'' BL21(DE3) are fermented in MJ9 medium <ref>Jansson M, Li YC, Jendeberg L, Anderson S, Montelione GT, Nilsson B (1996) High-level production of uniformly 15N- and 13C-enriched fusion proteins in ''Escherichia coli''. J Biomol NMR 7: 131-141</ref>. Cell pellets are stored at -20º C.

#add 30 ml Lysis buffer  to the a frozen cell pellet and thaw.
#sonicate in ice bath
#centrifuge to remove insoluble part
#supernatant is added to an AkTAxpressTM system with a His TrapHP column followed by HiLoad16/60 Superdex 75 gel filtration chromatography.
#exchange buffer to screening buffer by concentrating, diluting with new buffer, reconcentrating to 0.3 - 1.0 mM with Amicon ultrafiltration concentrator (Millipore). 

== Typical University of Toronto (Arrowsmith proteomics NMR lab) 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, 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 

 

The final NMR buffer for [U-15N, 13C]-labeled 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 ==

Required buffers:

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 

#add 25 mL of i015t8.5n500z into a frozen cell pellet and thaw. 
#sonicate in ice bath
#spin down cell pellet
#transfer supernatant into new falcon tube and add 3 mL of nickel beads
#rock the tube for at least 30 minutes in cold.
#spin down the beads and discard the supernatant
#wash the beads with i015t8.5n500z twice and with i030t8.5n500z twice 
#in the final i030t8.5n500z wash,  pour the beads unto gravity filter column 
#elute the protein with i500t8.5n500z
#add benzamidine, and add DTT 
#exchange buffer by concentrating, diluting with new buffer, reconcentrating in a vivaspin concentrator. 

if it is [U-15N, 13C]-labeled, 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

2011-05-03T13:53:24Z

TheresaRamelot:

== Samples for NMR ==

For each protein, we usually make the following samples:

*[''U''-15N, 13C]-labeled sample, for resonance assignment and NOE interpretation.
*
A U-15N and fractional 5%-13C-labeled sample for stereospecific assignment of Val and Leu isopropyl methyl groups based on the method of Neri et. al. (Biochemistry 28, 7510-7516,1989) that we call the NC5 sample.  This sample is prepared using 5% ''U''[1H,13C]-D-glucose and 95% unlabelled glucose in the ''E. coli'' minimal  growth media.

The NC5 sample is not [''U''-5%-13C,''U''-15N] since it is not uniformly 5% -13C labeled.  The incorporation of 13C in the methyl groups is biosynthetically directed by the ''E. coli''. 

This sample can be called: 

           <style> </style> ''<span style="font-size:11.0pt;mso-bidi-font-size:10.0pt;font-family:Helvetica;
mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman";
mso-ansi-language:EN-US;mso-fareast-language:EN-US">U''<span style="font-size:11.0pt;mso-bidi-font-size:10.0pt;font-family:Helvetica;
mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman";
mso-ansi-language:EN-US;mso-fareast-language:EN-US">-15N, 5%
biosynthetically directed 13C (NC5) sample or

            ''<span style="font-size:11.0pt;mso-bidi-font-size:10.0pt;font-family:Helvetica;
mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman";
mso-ansi-language:EN-US;mso-fareast-language:EN-US">U''-15N, fractional 5%-13C-labeled<span style="font-size:11.0pt;mso-bidi-font-size:10.0pt;font-family:Helvetica;
mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman";
mso-ansi-language:EN-US;mso-fareast-language:EN-US"> (NC5) sample  

and give the Neri reference.

 For RDC measurement:

*A secondary NC5 sample for RDC measurement. 

(Alternatively, for the H-N RDC measurement, a [U-15N]-labeled sample can be used. Rutger's uses a second NC5 sample, since this is all ready being prepared for the stereospecific assignments, and a 13C CHSQC can be compared with the [U-15N, 13C]-labeled sample, to check if the samples are the same.)

 For each protein that is a dimer in solution an extra sample may be required in addition to the samples above:

*1:1 mixture of natural abundance and [U-15N, 13C]-labeled samples, for intermolecular NOE interpretation.

 

== Typical Rutgers University NMR Buffers ==

The protein production facility at Rutgers University uses these three NMR buffers for the initial protein screening: <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 ==

''E.coli'' BL21(DE3) are fermented in MJ9 medium <ref>Jansson M, Li YC, Jendeberg L, Anderson S, Montelione GT, Nilsson B (1996) High-level production of uniformly 15N- and 13C-enriched fusion proteins in ''Escherichia coli''. J Biomol NMR 7: 131-141</ref>. Cell pellets are stored at -20º C.

#add 30 ml Lysis buffer  to the a frozen cell pellet and thaw.
#sonicate in ice bath
#centrifuge to remove insoluble part
#supernatant is added to an AkTAxpressTM system with a His TrapHP column followed by HiLoad16/60 Superdex 75 gel filtration chromatography.
#exchange buffer to screening buffer by concentrating, diluting with new buffer, reconcentrating to 0.3 - 1.0 mM with Amicon ultrafiltration concentrator (Millipore). 

== Typical University of Toronto (Arrowsmith proteomics NMR lab) 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, 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 

 

The final NMR buffer for [U-15N, 13C]-labeled 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 ==

Required buffers:

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 

#add 25 mL of i015t8.5n500z into a frozen cell pellet and thaw. 
#sonicate in ice bath
#spin down cell pellet
#transfer supernatant into new falcon tube and add 3 mL of nickel beads
#rock the tube for at least 30 minutes in cold.
#spin down the beads and discard the supernatant
#wash the beads with i015t8.5n500z twice and with i030t8.5n500z twice 
#in the final i030t8.5n500z wash,  pour the beads unto gravity filter column 
#elute the protein with i500t8.5n500z
#add benzamidine, and add DTT 
#exchange buffer by concentrating, diluting with new buffer, reconcentrating in a vivaspin concentrator. 

if it is [U-15N, 13C]-labeled, 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 />