Protein purification - Revision history

Bwu at 17:02, 8 March 2013

2013-03-08T17:02:21Z

← Older revision		Revision as of 17:02, 8 March 2013
Line 55:		Line 55:
	== Typical University of Toronto (Arrowsmith proteomics NMR lab) NMR Buffers ==		== Typical University of Toronto (Arrowsmith proteomics NMR lab) NMR Buffers ==

	~~<meta content="text/html; charset=utf~~-~~8" http~~-~~equiv="CONTENT-TYPE"><title></title> <meta content="OpenOffice.org 3.1 (Linux)" name="GENERATOR">~~<~~style type="text/css"><!--~~		'''15N-labelled Glucose-based autoinduction media''' : <br>
	~~@page { margin: 0.79in }~~
	~~P { margin-bottom~~: ~~0.08in }~~
	--> </style> <meta content="text/html; charset=utf-8" http-equiv="CONTENT-TYPE"><title></title> <meta content="OpenOffice.org 3.1 (Linux)" name="GENERATOR"><style type="text/css"><~~!--~~
	~~@page { margin: 0.79in }~~
	~~P { margin-bottom: 0.08in }~~
	--> ~~</style>~~

	~~'''15N-labelled Glucose-based autoinduction media''' : <br> </meta></meta>~~ This is for 15N-labelling of samples for screening so we can take advantage of autoinduction during the screening phase when we are dealing with a large number of samples, but be able to switch to IPTG-induction with <sup>13</sup>C-glucose as the sole carbon source for the smaller number of targets prepared for full NMR data collection, with high reproducibility. ~~<meta content="text~~/~~html; charset=utf~~-~~8" http-equiv="CONTENT-TYPE"~~>~~&lt~~;~~title&gt~~;~~&lt~~;/~~title&gt~~; ~~&lt~~;~~meta content="OpenOffice~~.~~org 3~~.~~1 (Linux)" name="GENERATOR"><style type="text/css"><!--~~		This is for 15N-labelling of samples for screening so we can take advantage of autoinduction during the screening phase when we are dealing with a large number of samples, but be able to switch to IPTG-induction with <sup>13</sup>C-glucose as the sole carbon source for the smaller number of targets prepared for full NMR data collection, with high reproducibility. <br>
	~~@page { margin: 0~~.~~79in }~~
	~~P { margin-bottom:~~ 0.~~08in }~~		For each sample, dissolve 6.8 g of Na<sub>2</sub>HPO<sub>4</sub>, 3g of KH<sub>2</sub>PO<sub>4</sub>, 0.5g of NaCl, and 0.6g of <sup>15</sup>N-labelled NH<sub>4</sub>Cl, in 500 mL water and autoclave the media.  <br>
	--> ~~<~~/~~style>~~
			In a separate small beaker or falcon tube, mix the following: 1 mL of 1M MgSO<sub>4</sub> stock solution; 5 mg of biotin; 5 mg of thiamine<sup>.</sup>HCl; 10 uL of 1M ZnSO<sub>4</sub> stock solution; 100 uL of 1M CaCl<sub>2</sub> stock solution; 2.5 g of glucose; 1 g of lactose and enough water to make 10 mL. Stir or shake until the sugar dissolves. Filter sterilize through a 0.2 micron syringe filter into the autoclaved <sup>15</sup>N-labelled solution.

	<br> For each sample, dissolve 6.8 g of Na<sub>2</sub>HPO<sub>4</sub>, 3g of KH<sub>2</sub>PO<sub>4</sub>, 0.5g of NaCl, and 0.6g of <sup>15</sup>N-labelled NH<sub>4</sub>Cl, in 500 mL water and autoclave the media.  <br> <meta content="text/html; charset=utf-8" http-equiv="CONTENT-TYPE"><title></title> <meta content="OpenOffice.org 3.1 (Linux)" name="GENERATOR"><style type="text/css"><!--
	~~@page { margin: 0.79in }~~
	~~P { margin-bottom: 0.08in }~~
	-->In a separate small beaker or falcon tube, mix the following: 1 mL of 1M MgSO<sub>4</sub> stock solution; 5 mg of biotin; 5 mg of thiamine<sup>.</sup>HCl; 10 uL of 1M ZnSO<sub>4</sub> stock solution; 100 uL of 1M CaCl<sub>2</sub> stock solution; 2.5 g of glucose; 1 g of lactose and enough water to make 10 mL. Stir or shake until the sugar dissolves. Filter sterilize through a 0.2 micron syringe filter into the autoclaved <sup>15</sup>N-labelled solution.</style></meta></meta> <meta content="text/html; charset=utf-8" http-equiv="CONTENT-TYPE"><title></title> <meta content="OpenOffice.org 3.1 (Linux)" name="GENERATOR"><style type="text/css"><!--
	~~@page { margin: 0.79in }~~
	~~P { margin-bottom: 0.08in }~~
	-->n a separate small beaker or falcon tube, mix the following: 1 mL of 1M MgSO<sub>4</sub> stock solution; 5 mg of biotin; 5 mg of thiamine<sup>.</sup>HCl; 10 uL of 1M ZnSO<sub>4</sub> stock solution; 100 uL of 1M CaCl<sub>2</sub> stock solution; 2.5 g of glucose; 1 g of lactose and enough water to make 10 mL. Stir or shake until the sugar dissolves. Filter sterilize through a 0.2 micron syringe filter into the autoclaved <sup>15</sup>N-labelled solution.</style></meta></meta></meta></meta></meta></meta> <meta content="text/html; charset=utf-8" http-equiv="CONTENT-TYPE"><title></title> <meta content="OpenOffice.org 3.1 (Linux)" name="GENERATOR"><style type="text/css"><!--
	~~@page { margin: 0.79in }~~
	~~P { margin-bottom: 0.08in }~~
	--> </style> In a separate small beaker or falcon tube, mix the following: 1 mL of 1M MgSO<sub>4</sub> stock solution; 5 mg of biotin; 5 mg of thiamine<sup>.</sup>HCl; 10 uL of 1M ZnSO<sub>4</sub> stock solution; 100 uL of 1M CaCl<sub>2</sub> stock solution; 2.5 g of glucose; 1 g of lactose and enough water to make 10 mL. Stir or shake until the sugar dissolves. Filter sterilize through a 0.2 micron syringe filter into the autoclaved <sup>15</sup>N-labelled solution.

	~~<br>~~

	Follow the same fermentation protocol as with IPTG-induction method, except there is no need to monitor the cell density and omit the addition of IPTG. Grow the cells at 37C throughout. &~~lt;/meta></meta&gt~~;		Follow the same fermentation protocol as with IPTG-induction method, except there is no need to monitor the cell density and omit the addition of IPTG. Grow the cells at 37C throughout.

	<br>		<br>
Line 150:		Line 134:
	The final NMR buffer for [U-<sup>15</sup>N, <sup>13</sup>C]-labeled sample depends on the protein of interest. all NMR buffers always contain : 0.01 % NaN<sub>3</sub>, 1 mM benzamidine, 1x inhibitor cocktail.		The final NMR buffer for [U-<sup>15</sup>N, <sup>13</sup>C]-labeled sample depends on the protein of interest. all NMR buffers always contain : 0.01 % NaN<sub>3</sub>, 1 mM benzamidine, 1x inhibitor cocktail.

	(1) If the protein has no cysteine in the sequence, do not bother to add ZnSO<sub>4</sub> and DTT (Zn ion will just be a nuisance and deuterated DTT is expensive).		(1) If the protein has no cysteine in the sequence, do not bother to add ZnSO<sub>4</sub> and DTT (Zn ion will just be a nuisance and deuterated DTT is expensive).

	== Typical University of Toronto Protein Purification Protocol<br> ==		== Typical University of Toronto Protein Purification Protocol<br> ==

Bwu at 22:21, 9 January 2013

2013-01-09T22:21:30Z

← Older revision		Revision as of 22:21, 9 January 2013
Line 54:		Line 54:

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

			<meta content="text/html; charset=utf-8" http-equiv="CONTENT-TYPE"><title></title> <meta content="OpenOffice.org 3.1 (Linux)" name="GENERATOR"><style type="text/css"><!--
			@page { margin: 0.79in }
			P { margin-bottom: 0.08in }
			--> </style> <meta content="text/html; charset=utf-8" http-equiv="CONTENT-TYPE"><title></title> <meta content="OpenOffice.org 3.1 (Linux)" name="GENERATOR"><style type="text/css"><!--
			@page { margin: 0.79in }
			P { margin-bottom: 0.08in }
			--> </style>

			'''15N-labelled Glucose-based autoinduction media''' : <br> </meta></meta> This is for 15N-labelling of samples for screening so we can take advantage of autoinduction during the screening phase when we are dealing with a large number of samples, but be able to switch to IPTG-induction with <sup>13</sup>C-glucose as the sole carbon source for the smaller number of targets prepared for full NMR data collection, with high reproducibility. <meta content="text/html; charset=utf-8" http-equiv="CONTENT-TYPE"><title></title> <meta content="OpenOffice.org 3.1 (Linux)" name="GENERATOR"><style type="text/css"><!--
			@page { margin: 0.79in }
			P { margin-bottom: 0.08in }
			--> </style>

			<br> For each sample, dissolve 6.8 g of Na<sub>2</sub>HPO<sub>4</sub>, 3g of KH<sub>2</sub>PO<sub>4</sub>, 0.5g of NaCl, and 0.6g of <sup>15</sup>N-labelled NH<sub>4</sub>Cl, in 500 mL water and autoclave the media.  <br> <meta content="text/html; charset=utf-8" http-equiv="CONTENT-TYPE"><title></title> <meta content="OpenOffice.org 3.1 (Linux)" name="GENERATOR"><style type="text/css"><!--
			@page { margin: 0.79in }
			P { margin-bottom: 0.08in }
			-->In a separate small beaker or falcon tube, mix the following: 1 mL of 1M MgSO<sub>4</sub> stock solution; 5 mg of biotin; 5 mg of thiamine<sup>.</sup>HCl; 10 uL of 1M ZnSO<sub>4</sub> stock solution; 100 uL of 1M CaCl<sub>2</sub> stock solution; 2.5 g of glucose; 1 g of lactose and enough water to make 10 mL. Stir or shake until the sugar dissolves. Filter sterilize through a 0.2 micron syringe filter into the autoclaved <sup>15</sup>N-labelled solution.</style></meta></meta> <meta content="text/html; charset=utf-8" http-equiv="CONTENT-TYPE"><title></title> <meta content="OpenOffice.org 3.1 (Linux)" name="GENERATOR"><style type="text/css"><!--
			@page { margin: 0.79in }
			P { margin-bottom: 0.08in }
			-->n a separate small beaker or falcon tube, mix the following: 1 mL of 1M MgSO<sub>4</sub> stock solution; 5 mg of biotin; 5 mg of thiamine<sup>.</sup>HCl; 10 uL of 1M ZnSO<sub>4</sub> stock solution; 100 uL of 1M CaCl<sub>2</sub> stock solution; 2.5 g of glucose; 1 g of lactose and enough water to make 10 mL. Stir or shake until the sugar dissolves. Filter sterilize through a 0.2 micron syringe filter into the autoclaved <sup>15</sup>N-labelled solution.</style></meta></meta></meta></meta></meta></meta> <meta content="text/html; charset=utf-8" http-equiv="CONTENT-TYPE"><title></title> <meta content="OpenOffice.org 3.1 (Linux)" name="GENERATOR"><style type="text/css"><!--
			@page { margin: 0.79in }
			P { margin-bottom: 0.08in }
			--> </style> In a separate small beaker or falcon tube, mix the following: 1 mL of 1M MgSO<sub>4</sub> stock solution; 5 mg of biotin; 5 mg of thiamine<sup>.</sup>HCl; 10 uL of 1M ZnSO<sub>4</sub> stock solution; 100 uL of 1M CaCl<sub>2</sub> stock solution; 2.5 g of glucose; 1 g of lactose and enough water to make 10 mL. Stir or shake until the sugar dissolves. Filter sterilize through a 0.2 micron syringe filter into the autoclaved <sup>15</sup>N-labelled solution.

			<br>

			Follow the same fermentation protocol as with IPTG-induction method, except there is no need to monitor the cell density and omit the addition of IPTG. Grow the cells at 37C throughout. </meta></meta>

			<br>

	Standard screening NMR buffers are:		Standard screening NMR buffers are:

			<br>

	{\| cellspacing="1" cellpadding="1" border="1" style="width: 548px; height: 238px;"		{\| cellspacing="1" cellpadding="1" border="1" style="width: 548px; height: 238px;"
	\|-		\|-
	\| name		\| name
	\| buffer		\| buffer
	\| NaCl concentration		\| NaCl concentration
	\| pH		\| pH
	\|-		\|-
	\| A5.0n300zd		\| A5.0n300zd
	\| Sodium acetate		\| Sodium acetate
	\| 300 mM		\| 300 mM
	\| 5.0		\| 5.0
	\|-		\|-
	\| Bt6.0n450zd		\| Bt6.0n450zd
	\| bis-tris		\| bis-tris
	\| 450 mM		\| 450 mM
	\| 6.0		\| 6.0
	\|-		\|-
	\| Bt6.5n450zd		\| Bt6.5n450zd
	\| bis-tris		\| bis-tris
	\| 450 mM		\| 450 mM
	\| 6.5		\| 6.5
	\|-		\|-
	\| M6.5n450zd		\| M6.5n450zd
	\| MOPS		\| MOPS
	\| 450 mM		\| 450 mM
	\| 6.5		\| 6.5
	\|-		\|-
	\| P6.5n450zd		\| P6.5n450zd
	\| Sodium phosphate		\| Sodium phosphate
	\| 450 mM		\| 450 mM
	\| 6.5		\| 6.5
	\|-		\|-
	\| P7.0n450zd		\| P7.0n450zd
	\| Sodium phosphate		\| Sodium phosphate
	\| 450 mM		\| 450 mM
	\| 7.0		\| 7.0
	\|-		\|-
	\| H7.0n450zd		\| H7.0n450zd
	\| HEPES		\| HEPES
	\| 450 mM		\| 450 mM
	\| 7.0		\| 7.0
	\|-		\|-
	\| H7.5n450zd		\| H7.5n450zd
	\| HEPES		\| HEPES
	\| 450 mM		\| 450 mM
	\| 7.5		\| 7.5
	\|-		\|-
	\| T7.7n500zd		\| T7.7n500zd
	\| tris		\| tris
	\| 500 mM		\| 500 mM
	\| 7.7		\| 7.7
	\|}		\|}

			<br>

	All standard screening buffers contain 10 mM of the buffer, 10 uM ZnSO4, 10 mM DTT, 0.01% NaN3, 1 mM benzamidine, 1x inhibitor cocktail, 5% D2O.<br>		All standard screening buffers contain 10 mM of the buffer, 10 uM ZnSO4, 10 mM DTT, 0.01% NaN3, 1 mM benzamidine, 1x inhibitor cocktail, 5% D2O.<br>

			<br>

	The final NMR buffer for [U-<sup>15</sup>N, <sup>13</sup>C]-labeled sample depends on the protein of interest. all NMR buffers always contain : 0.01 % NaN<sub>3</sub>, 1 mM benzamidine, 1x inhibitor cocktail.		The final NMR buffer for [U-<sup>15</sup>N, <sup>13</sup>C]-labeled sample depends on the protein of interest. all NMR buffers always contain : 0.01 % NaN<sub>3</sub>, 1 mM benzamidine, 1x inhibitor cocktail.

Bwu at 20:48, 5 October 2011

2011-10-05T20:48:48Z

@@ Line 57: / Line 57: @@
 Standard screening NMR buffers are:
 <br>
 The final NMR buffer for [U-<sup>15</sup>N, <sup>13</sup>C]-labeled sample depends on the protein of interest. all NMR buffers always contain&nbsp;: 0.01&nbsp;%&nbsp;NaN<sub>3</sub>, 1 mM&nbsp;benzamidine, 1x inhibitor cocktail.

TheresaRamelot at 13:54, 3 May 2011

2011-05-03T13:54:04Z

← Older revision		Revision as of 13:54, 3 May 2011
Line 5:		Line 5:
	*[''U''-<sup>15</sup>N, <sup>13</sup>C]-labeled sample, for resonance assignment and NOE interpretation.		*[''U''-<sup>15</sup>N, <sup>13</sup>C]-labeled sample, for resonance assignment and NOE interpretation.
	*		*
	A U-<sup>15</sup>N and fractional 5%-<sup>13</sup>C-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''[<sup>1</sup>H,<sup>13</sup>C]-D-glucose and 95% unlabelled glucose in the ''E. coli'' minimal  growth media.

			A U-<sup>15</sup>N and fractional 5%-<sup>13</sup>C-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''[<sup>1</sup>H,<sup>13</sup>C]-D-glucose and 95% unlabelled glucose in the ''E. coli'' minimal  growth media.

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

	This sample can be called:<br>		This sample can be called:<br>

	<style><!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0in; margin-bottom:.0001pt; text-align:justify; mso-pagination:widow-orphan; font-size:11.0pt; mso-bidi-font-size:10.0pt; font-family:"Times New Roman"; mso-ascii-font-family:Helvetica; mso-fareast-font-family:"Times New Roman"; mso-hansi-font-family:Helvetica; mso-bidi-font-family:"Times New Roman";} @page Section1 {size:8.5in 11.0in; margin:1.0in 1.25in 1.0in 1.25in; mso-header-margin:.5in; mso-footer-margin:.5in; mso-paper-source:0;} div.Section1 {page:Section1;} --> </style> ''<span ~~style="font-size:11.0pt;mso-bidi-font-size:10.0pt;font-family:Helvetica;~~		''<span>U</span>''<span>-<sup>15</sup>N, 5%
	~~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>''<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"~~>-<sup>15</sup>N, 5%
	biosynthetically directed <sup>13</sup>C (NC5) sample or</span>		biosynthetically directed <sup>13</sup>C (NC5) sample or</span>

	''<span ~~style="font-size:11.0pt;mso-bidi-font-size:10.0pt;font-family:Helvetica;~~		''<span>U</span>''<span style="font-size: 11pt; font-family: Helvetica;">-<sup>15</sup>N, fractional</span> 5%-<sup>13</sup>C-labeled<span> (NC5) sample </span><span style="font-size: 11pt; font-family: Helvetica;"> </span>
	~~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>''<span style="font-size: 11pt; font-family: Helvetica;">-<sup>15</sup>N, fractional</span> 5%-<sup>13</sup>C-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 </span><span style="font-size: 11pt; font-family: Helvetica;"> </span>

	<span style="font-size: 11pt; font-family: Helvetica;">and give the Neri reference.</span>		<span style="font-size: 11pt; font-family: Helvetica;">and give the Neri reference.</span>

	<br> For RDC measurement:		<br> For RDC measurement:

TheresaRamelot at 13:53, 3 May 2011

2011-05-03T13:53:24Z

@@ Line 3: / Line 3: @@
 For each protein, we usually make the following samples:
-*[U-<sup>15</sup>N, <sup>13</sup>C]-labeled sample, for resonance assignment and NOE interpretation.
+*[''U''-<sup>15</sup>N, <sup>13</sup>C]-labeled sample, for resonance assignment and NOE interpretation.
 *A U-<sup>15</sup>N and fractional 5%-<sup>13</sup>C-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.&nbsp; This sample is prepared using 5% U[<sup>1</sup>H,<sup>13</sup>C]-D-glucose and 95% unlabelled glucose in the ''E. coli'' minimal&nbsp; growth media.<br>
 <br> For RDC measurement:

TheresaRamelot at 16:27, 10 March 2011

2011-03-10T16:27:39Z

← Older revision		Revision as of 16:27, 10 March 2011
Line 4:		Line 4:

	*[U-<sup>15</sup>N, <sup>13</sup>C]-labeled sample, for resonance assignment and NOE interpretation.		*[U-<sup>15</sup>N, <sup>13</sup>C]-labeled sample, for resonance assignment and NOE interpretation.
	*A U-<sup>15</sup>N and fractional 5%-<sup>13</sup>C-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[<sup>1</sup>H,<sup>13</sup>C]-D-glucose and 95% unlabelled glucose in the ''E. coli'' minimal  growth media.<br>		*A U-<sup>15</sup>N and fractional 5%-<sup>13</sup>C-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[<sup>1</sup>H,<sup>13</sup>C]-D-glucose and 95% unlabelled glucose in the ''E. coli'' minimal  growth media.<br>

	<br> For RDC measurement:		<br> For RDC measurement:

TheresaRamelot at 16:26, 10 March 2011

2011-03-10T16:26:08Z

Alex: /* Typical University of Toronto Protein Purification Protocol */

2009-12-16T02:33:41Z

Typical University of Toronto Protein Purification Protocol

@@ Line 67: / Line 67: @@
 M&nbsp;benzamidine <br> <br>
-(1) add 25 mL of i015t8.5n500z into a frozen cell pellet and thaw.<br>
+#add 25 mL of i015t8.5n500z into a frozen cell pellet and thaw.<br>
-(2) sonicate in ice bath
+if it is [U-<sup>15</sup>N, <sup>13</sup>C]-labeled, add step
 (9a) put sample in dialysis bag with protease to cut his-tag and dialyse&nbsp; against&nbsp; cleavage buffer

Alex: /* Typical University of Toronto (Arrowsmith proteomics NMR lab) NMR Buffers */

2009-12-16T02:31:30Z

Typical University of Toronto (Arrowsmith proteomics NMR lab) NMR Buffers

← Older revision		Revision as of 02:31, 16 December 2009
Line 42:		Line 42:
	== Typical University of Toronto (Arrowsmith proteomics NMR lab) NMR Buffers ==		== Typical University of Toronto (Arrowsmith proteomics NMR lab) NMR Buffers ==

	Standard screening NMR buffers are: ~~<br> <br>~~ a5.0n300zd : 10 mM sodium acetate, pH 5.0, 300 mM NaCl, 10 uM ZnSO<sub>4</sub>, 10 mM DTT, 0.01 % NaN<sub>3</sub>, 1 mM benzamidine, 1x inhibitor cocktail, 5% D<sub>2</sub>O<br>		Standard screening NMR buffers are:
			*a5.0n300zd : 10 mM sodium acetate, pH 5.0, 300 mM NaCl, 10 uM ZnSO<sub>4</sub>, 10 mM DTT, 0.01 % NaN<sub>3</sub>, 1 mM benzamidine, 1x inhibitor cocktail, 5% D<sub>2</sub>O<br>
	m6.5n450zd: 10 mM MOPS, pH 6.5, 450 mM NaCl, 10 uM ZnSO<sub>4</sub>, 10 mM DTT, 0.01 % NaN<sub>3</sub>, 1 mM benzamidine, 1x inhibitor cocktail, 5% D<sub>2</sub>O		*m6.5n450zd: 10 mM MOPS, pH 6.5, 450 mM NaCl, 10 uM ZnSO<sub>4</sub>, 10 mM DTT, 0.01 % NaN<sub>3</sub>, 1 mM benzamidine, 1x inhibitor cocktail, 5% D<sub>2</sub>O
			*t7.7n500zd : 10 mM tris, pH 7.7, 500 mM NaCl, 10 uM ZnSO<sub>4</sub>, 10 mM DTT, 0.01 % NaN<sub>3</sub>, 1 mM benzamidine, 1x inhibitor cocktail, 5% D<sub>2</sub>O<br>
	t7.7n500zd : 10 mM tris, pH 7.7, 500 mM NaCl, 10 uM ZnSO<sub>4</sub>, 10 mM DTT, 0.01 % NaN<sub>3</sub>, 1 mM benzamidine, 1x inhibitor cocktail, 5% D<sub>2</sub>O<br>

	<br>		<br>

	The final ~~nmr~~ buffer for <sup>13</sup>C/<sup>15</sup>~~N labelled~~ sample depends on the protein of interest. all ~~nmr~~ buffers always contain : 0.01 % NaN<sub>3</sub>, 1 mM benzamidine, 1x inhibitor cocktail.		The final NMR buffer for [U-<sup>15</sup>N, <sup>13</sup>C]-labeled sample depends on the protein of interest. all NMR buffers always contain : 0.01 % NaN<sub>3</sub>, 1 mM benzamidine, 1x inhibitor cocktail.

	(1) If the protein has no cysteine in the sequence, do not bother to add ZnSO<sub>4</sub> and DTT (Zn ion will just be a nuisance and deuterated DTT is expensive).		(1) If the protein has no cysteine in the sequence, do not bother to add ZnSO<sub>4</sub> and DTT (Zn ion will just be a nuisance and deuterated DTT is expensive).

Alex: /* Typical Rutgers University Protein Purification Protocol */

2009-12-16T02:29:22Z

Typical Rutgers University Protein Purification Protocol

← Older revision		Revision as of 02:29, 16 December 2009
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	== Typical Rutgers University Protein Purification Protocol<br> ==		== Typical Rutgers University Protein Purification Protocol<br> ==

	''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>.		''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.

	~~Cell pellets are stored at -20º C.~~		#add <!--StartFragment--><font face="Verdana, Helvetica, Arial"><span style="font-size: 12px;">30 ml Lysis buffer</span></font> <!--EndFragment--> to the a frozen cell pellet and thaw.
			#sonicate in ice bath
	~~(1)~~ add <!--StartFragment--><font face="Verdana, Helvetica, Arial"><span style="font-size: 12px;">30 ml Lysis buffer</span></font> <!--EndFragment--> to the a frozen cell pellet and thaw.		#centrifuge to remove insoluble part
			#supernatant is added to an AkTAxpress<sup>TM</sup> system with a His TrapHP column followed by HiLoad16/60 Superdex 75 gel filtration chromatography.
	~~(2)~~ sonicate in ice bath		#exchange buffer to screening buffer by concentrating, diluting with new buffer, reconcentrating to 0.3 - 1.0 mM with Amicon ultrafiltration concentrator (Millipore). <br><br>

	~~(3)~~ centrifuge to remove insoluble part

	~~(4)~~ supernatant is added to an AkTAxpress<sup>TM</sup> system with a His TrapHP column followed by HiLoad16/60 Superdex 75 gel filtration chromatography.

	~~(5)~~ exchange buffer to screening buffer by concentrating, diluting with new buffer, reconcentrating to 0.3 - 1.0 mM with Amicon ultrafiltration concentrator (Millipore). <br><br>

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

← Older revision		Revision as of 16:26, 10 March 2011
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	*[U-<sup>15</sup>N, <sup>13</sup>C]-labeled sample, for resonance assignment and NOE interpretation.		*[U-<sup>15</sup>N, <sup>13</sup>C]-labeled sample, for resonance assignment and NOE interpretation.
	*[U-<sup>15</sup>N, 5%-<sup>13</sup>C]-labeled sample, for stereospecific assignment of ~~VAL~~ and ~~LEU~~ isopropyl ~~moieties~~.		*A U-<sup>15</sup>N and fractional 5%-<sup>13</sup>C-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[<sup>1</sup>H,<sup>13</sup>C]-D-glucose and 95% unlabelled glucose in the ''E. coli'' minimal  growth media.<br>

	<br> For RDC measurement:		<br> For RDC measurement:

	*A secondary ~~[U-<sup>15</sup>N, 5%-<sup>13</sup>C]-labeled~~ sample, for RDC measurement.<br>		*A secondary NC5 sample for RDC measurement.<br>

	(Alternatively, for the H-N RDC measurement, a [U-<sup>15</sup>N]-labeled sample can be used. Rutger's uses a second ~~[U-<sup>15</sup>N, 5%-<sup>13</sup>C]-labeled~~ sample, since this is all ready being prepared for the stereospecific assignments, and a <sup>13</sup>C CHSQC can be compared with the [U-<sup>15</sup>N, <sup>13</sup>C]-labeled sample, to check if the samples are the same.)		(Alternatively, for the H-N RDC measurement, a [U-<sup>15</sup>N]-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 <sup>13</sup>C CHSQC can be compared with the [U-<sup>15</sup>N, <sup>13</sup>C]-labeled sample, to check if the samples are the same.)

	<br> For each protein that is a dimer in solution an extra sample may be required in addition to the samples above:		<br> 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-<sup>15</sup>N, <sup>13</sup>C]-labeled samples, for intermolecular NOE interpretation.		*1:1 mixture of natural abundance and [U-<sup>15</sup>N, <sup>13</sup>C]-labeled samples, for intermolecular NOE interpretation.

	<br>		<br>

	== Typical Rutgers University NMR Buffers<br> ==		== Typical Rutgers University NMR Buffers<br> ==
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	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>:<br>		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>:<br>

			<br>

	*pH 4.5 NMR buffer:<span style="">  </span>20 mM NH<sub>4</sub>OAc, 100 mM NaCl, 10 mM DTT, 5 mM CaCl<sub>2</sub>, 0.02% NaN<sub>3</sub>, 5% D<sub>2</sub>O		*pH 4.5 NMR buffer:<span style="">  </span>20 mM NH<sub>4</sub>OAc, 100 mM NaCl, 10 mM DTT, 5 mM CaCl<sub>2</sub>, 0.02% NaN<sub>3</sub>, 5% D<sub>2</sub>O
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	*pH 6.5 NMR buffer:<span style="">  </span>20 mM MES, 100 mM NaCl, 5 mM CaCl<sub>2</sub>, 10 mM DTT, 0.02% NaN<sub>3</sub>, 5% D<sub>2</sub>O		*pH 6.5 NMR buffer:<span style="">  </span>20 mM MES, 100 mM NaCl, 5 mM CaCl<sub>2</sub>, 10 mM DTT, 0.02% NaN<sub>3</sub>, 5% D<sub>2</sub>O

	<br>		<br>

	== Typical Rutgers University Protein Purification Protocol<br> ==		== Typical Rutgers University Protein Purification Protocol<br> ==
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	Standard screening NMR buffers are:		Standard screening NMR buffers are:

	*a5.0n300zd : 10 mM sodium acetate, pH 5.0, 300 mM NaCl, 10 uM ZnSO<sub>4</sub>, 10 mM DTT, 0.01 % NaN<sub>3</sub>, 1 mM benzamidine, 1x inhibitor cocktail, 5% D<sub>2</sub>O<br>		*a5.0n300zd : 10 mM sodium acetate, pH 5.0, 300 mM NaCl, 10 uM ZnSO<sub>4</sub>, 10 mM DTT, 0.01 % NaN<sub>3</sub>, 1 mM benzamidine, 1x inhibitor cocktail, 5% D<sub>2</sub>O<br>
	*m6.5n450zd: 10 mM MOPS, pH 6.5, 450 mM NaCl, 10 uM ZnSO<sub>4</sub>, 10 mM DTT, 0.01 % NaN<sub>3</sub>, 1 mM benzamidine, 1x inhibitor cocktail, 5% D<sub>2</sub>O		*m6.5n450zd: 10 mM MOPS, pH 6.5, 450 mM NaCl, 10 uM ZnSO<sub>4</sub>, 10 mM DTT, 0.01 % NaN<sub>3</sub>, 1 mM benzamidine, 1x inhibitor cocktail, 5% D<sub>2</sub>O
	*t7.7n500zd : 10 mM tris, pH 7.7, 500 mM NaCl, 10 uM ZnSO<sub>4</sub>, 10 mM DTT, 0.01 % NaN<sub>3</sub>, 1 mM benzamidine, 1x inhibitor cocktail, 5% D<sub>2</sub>O<br>		*t7.7n500zd : 10 mM tris, pH 7.7, 500 mM NaCl, 10 uM ZnSO<sub>4</sub>, 10 mM DTT, 0.01 % NaN<sub>3</sub>, 1 mM benzamidine, 1x inhibitor cocktail, 5% D<sub>2</sub>O<br>

	<br>		<br>
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	The final NMR buffer for [U-<sup>15</sup>N, <sup>13</sup>C]-labeled sample depends on the protein of interest. all NMR buffers always contain : 0.01 % NaN<sub>3</sub>, 1 mM benzamidine, 1x inhibitor cocktail.		The final NMR buffer for [U-<sup>15</sup>N, <sup>13</sup>C]-labeled sample depends on the protein of interest. all NMR buffers always contain : 0.01 % NaN<sub>3</sub>, 1 mM benzamidine, 1x inhibitor cocktail.

	(1) If the protein has no cysteine in the sequence, do not bother to add ZnSO<sub>4</sub> and DTT (Zn ion will just be a nuisance and deuterated DTT is expensive).		(1) If the protein has no cysteine in the sequence, do not bother to add ZnSO<sub>4</sub> and DTT (Zn ion will just be a nuisance and deuterated DTT is expensive).

	== Typical University of Toronto Protein Purification Protocol<br> ==		== Typical University of Toronto Protein Purification Protocol<br> ==
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	#elute the protein with i500t8.5n500z		#elute the protein with i500t8.5n500z
	#add benzamidine, and add DTT<br>		#add benzamidine, and add DTT<br>
	#exchange buffer by concentrating, diluting with new buffer, reconcentrating in a vivaspin concentrator.<br> <br>		#exchange buffer by concentrating, diluting with new buffer, reconcentrating in a vivaspin concentrator.<br> <br>

	if it is [U-<sup>15</sup>N, <sup>13</sup>C]-labeled, add step		if it is [U-<sup>15</sup>N, <sup>13</sup>C]-labeled, add step
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	(9a) put sample in dialysis bag with protease to cut his-tag and dialyse  against  cleavage buffer		(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. <br>		(9b) pass the sample through nickel beads again, then follow step (10) above. <br>

	== References ==		== References ==

	<references />		<references />