ISISBio:Protocols/Purification/Tus - Revision history

Christopher C Vanlang at 01:54, 30 December 2011

2011-12-30T01:54:39Z

←Older revision		Revision as of 01:54, 30 December 2011
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	==Overview==		==Overview==

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	<!-- You can tag this protocol with various categories. See the [[Categories]] page for more information. -->		<!-- You can tag this protocol with various categories. See the [[Categories]] page for more information. -->
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-	~~[[Category:Needs attention]]~~
	[[Category:Protein]]		[[Category:Protein]]
	[[Category:Purification]]		[[Category:Purification]]
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	<!-- Move the relevant categories above this line to tag your protocol with the label		<!-- Move the relevant categories above this line to tag your protocol with the label

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	[[Category:In vitro]]		[[Category:In vitro]]

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	[[Category:RNA]]		[[Category:RNA]]
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Cameron Neylon at 15:14, 1 August 2008

2008-08-01T15:14:44Z

←Older revision		Revision as of 15:14, 1 August 2008
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-	~~Cell Lysis~~

		+	==Overview==
		+
		+	Lysis and purification of the E. coli replication termination protein, Tus
		+
		+	==Materials==
		+	List reagents, supplies and equipment necessary to perform the protocol here. For those materials which have their own OWW pages, link to that page. Alternatively, links to the suppliers' page on that material are also appropriate.
		+
		+	*supply 1 (i.e. tubes of a certain size? spreaders?)
		+	*reagent 1
		+	*X μL reagent 2
		+	**component A (reagent 2 is made up of multiple components)
		+	**component B
		+	*equipment 1
		+	*equipment 2
		+
		+	==Procedure==
		+	===Lysis===
	The stored frozen cells Rosetta 2 pLysS/pCM862 were thawed at 4 ºC and resuspended in 15 mL.g-1 of ice-cold lysis buffer (50 mM Imidazole, pH 6.8, 1 mM EDTA 10% w/v sucrose, 0.05 M NaCl, 2 mM DTT). Lysozyme was added to a concentration of 0.2 mg.mL-1 and the suspension was stirred on ice for 30 minutes. The cells were then subjected to three freeze-thaw cycles, by freezing at -80 ºC and then thawing under cold running water. Cell debris was removed from the suspension by centrifugation (30 100 g, 10 minutes, 4 ºC).		The stored frozen cells Rosetta 2 pLysS/pCM862 were thawed at 4 ºC and resuspended in 15 mL.g-1 of ice-cold lysis buffer (50 mM Imidazole, pH 6.8, 1 mM EDTA 10% w/v sucrose, 0.05 M NaCl, 2 mM DTT). Lysozyme was added to a concentration of 0.2 mg.mL-1 and the suspension was stirred on ice for 30 minutes. The cells were then subjected to three freeze-thaw cycles, by freezing at -80 ºC and then thawing under cold running water. Cell debris was removed from the suspension by centrifugation (30 100 g, 10 minutes, 4 ºC).

	The volume of the supernatant was measured. The NaCl concentration was adjusted to 1M and solid ammonium sulfate to a final concentration of 0.078 g.mL<sup>-1</sup> slowly added. The resulting suspension was stirred on ice for 1 hour and then centrifuged again (30 100 g, 20 minutes, 4ºC). The supernatant was retained and a sample was taken for SDS-PAGE analysis. The volume of the supernatant was again measured and solid ammonium sulfate was slowly add a further 0.29 g.mL<sup>-1</sup>. The resulting suspension was stirred on ice for 2 hours before further centrifugation (30 100 g, 20 minutes, 1º C). A small sample of the supernatant was retained for SDS-PAGE, but the remainder was discarded and the pellet was resuspended in a minimal volume (~1-5 mL) of Buffer A (50 mM imidazole-HCl, pH 6.8, 1 mM EDTA, 1 mM DTT). The suspension was then transferred to 10-12 kDa cut off dialysis tubing and dialysed against two changes of Buffer A (at least 1 litre each for at least one hour).		The volume of the supernatant was measured. The NaCl concentration was adjusted to 1M and solid ammonium sulfate to a final concentration of 0.078 g.mL<sup>-1</sup> slowly added. The resulting suspension was stirred on ice for 1 hour and then centrifuged again (30 100 g, 20 minutes, 4ºC). The supernatant was retained and a sample was taken for SDS-PAGE analysis. The volume of the supernatant was again measured and solid ammonium sulfate was slowly add a further 0.29 g.mL<sup>-1</sup>. The resulting suspension was stirred on ice for 2 hours before further centrifugation (30 100 g, 20 minutes, 1º C). A small sample of the supernatant was retained for SDS-PAGE, but the remainder was discarded and the pellet was resuspended in a minimal volume (~1-5 mL) of Buffer A (50 mM imidazole-HCl, pH 6.8, 1 mM EDTA, 1 mM DTT). The suspension was then transferred to 10-12 kDa cut off dialysis tubing and dialysed against two changes of Buffer A (at least 1 litre each for at least one hour).

-	Purification	+	===Purification===

	The dialysed solution was diluted in Buffer A to an OD280 of ~2 and loaded onto a Pharmacia DEAE FastFlow (5mL) pre-packed column equilibrated in Buffer A. The column was washed with at least three column volumes of Buffer A and the Tus protein was eluted in a gradient of 0.00 to 1.50 M NaCl in 10 column volumes in Buffer A ####TUS-LPETGG-His6 MAY NOT ELUTE WHERE WE EXPECT - MAY BE IN FLOW THROUGH####. The fractions containing Tus were combined and loaded directly onto a 5-15 mL Whatmann P11 phosphocellulose column equilibrated in Buffer A. The column was washed with 5 column volumes of Buffer A and the bound protein was eluted by the application of a gradient from 0.00 to 1.50 M NaCl in Buffer A as before.		The dialysed solution was diluted in Buffer A to an OD280 of ~2 and loaded onto a Pharmacia DEAE FastFlow (5mL) pre-packed column equilibrated in Buffer A. The column was washed with at least three column volumes of Buffer A and the Tus protein was eluted in a gradient of 0.00 to 1.50 M NaCl in 10 column volumes in Buffer A ####TUS-LPETGG-His6 MAY NOT ELUTE WHERE WE EXPECT - MAY BE IN FLOW THROUGH####. The fractions containing Tus were combined and loaded directly onto a 5-15 mL Whatmann P11 phosphocellulose column equilibrated in Buffer A. The column was washed with 5 column volumes of Buffer A and the bound protein was eluted by the application of a gradient from 0.00 to 1.50 M NaCl in Buffer A as before.

	The resultant protein was exchanged into storage buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 20% w/v glycerol, 1 mM DTT) by dialysis (at least three times >100 volumes) or with a desalting column equilibrated in Storage buffer. Following exchange the protein was concentrated to ~10-100 μM using a spin concentrator or Amicon. The protein concentration was estimated from the UV Absorbance spectrum of the protein solution, and the samples were aliquoted and snap-frozen in liquid nitrogen before moving to store at –80 ºC.		The resultant protein was exchanged into storage buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 20% w/v glycerol, 1 mM DTT) by dialysis (at least three times >100 volumes) or with a desalting column equilibrated in Storage buffer. Following exchange the protein was concentrated to ~10-100 μM using a spin concentrator or Amicon. The protein concentration was estimated from the UV Absorbance spectrum of the protein solution, and the samples were aliquoted and snap-frozen in liquid nitrogen before moving to store at –80 ºC.
		+
		+
		+	==Notes==
		+	Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
		+	#List troubleshooting tips here.
		+	#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
		+	#Anecdotal observations that might be of use to others can also be posted here.
		+
		+	Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
		+
		+	==References==
		+	'''Relevant papers and books'''
		+	<!-- If this protocol has papers or books associated with it, list those references here. See the [[OpenWetWare:Biblio]] page for more information. -->
		+	<biblio>
		+
		+	</biblio>
		+
		+	==Contact==
		+	*'''[[User:Cameron Neylon\|Cameron Neylon]] 15:14, 1 August 2008 (UTC)''':
		+
		+	<!-- You can tag this protocol with various categories. See the [[Categories]] page for more information. -->
		+
		+	[[Category:Needs attention]]
		+	[[Category:Protein]]
		+	[[Category:Purification]]
		+	[[Category:Protocol]]
		+	[[Category:ISISBio]]
		+
		+	<!-- Move the relevant categories above this line to tag your protocol with the label
		+
		+
		+
		+
		+	[[Category:In vitro]]
		+
		+	[[Category:In vivo]]
		+
		+	[[Category:In silico]]
		+
		+	[[Category:DNA]]
		+
		+	[[Category:RNA]]
		+
		+
		+
		+	[[Category:Chemical]]
		+
		+	[[Category:Escherichia coli]]
		+
		+	[[Category:Yeast]]
		+	-->

Cameron Neylon at 13:26, 21 July 2008

2008-07-21T13:26:26Z

←Older revision		Revision as of 13:26, 21 July 2008
Line 1:		Line 1:
	Cell Lysis		Cell Lysis

-	The stored frozen cells Rosetta 2 pLysS/pCM862 were thawed at 4 ºC and resuspended in 15 mL.g-1 of ice-cold lysis buffer (50 mM Imidazole, pH 6.8, 1 mM EDTA 10% w/v sucrose, 0.05 M NaCl, 2 mM DTT~~, 10 mM spermidine-3HCl~~). Lysozyme was added to a concentration of 0.2 mg.mL-1 and the suspension was stirred on ice for 30 minutes. The cells were then subjected to three freeze-thaw cycles, by ~~direct addition of liquid nitrogen, whilst stirring until frozen~~ and then ~~fully defrosting at room temperature, whilst stirring~~. Cell debris was removed from the suspension by centrifugation (30 100 g, 10 minutes, 4 ºC).	+	The stored frozen cells Rosetta 2 pLysS/pCM862 were thawed at 4 ºC and resuspended in 15 mL.g-1 of ice-cold lysis buffer (50 mM Imidazole, pH 6.8, 1 mM EDTA 10% w/v sucrose, 0.05 M NaCl, 2 mM DTT). Lysozyme was added to a concentration of 0.2 mg.mL-1 and the suspension was stirred on ice for 30 minutes. The cells were then subjected to three freeze-thaw cycles, by freezing at -80 ºC and then thawing under cold running water. Cell debris was removed from the suspension by centrifugation (30 100 g, 10 minutes, 4 ºC).

-	The volume of the supernatant was measured. The NaCl concentration was adjusted to 1M and solid ammonium sulfate to a final concentration of ~~15%~~ slowly added. The resulting suspension was stirred on ice for 1 hour and then centrifuged again (30 100 g, 20 minutes, 4ºC). The supernatant was retained and a sample was taken for SDS-PAGE analysis. The volume of the supernatant was again measured and solid ammonium sulfate was slowly ~~added to~~ a ~~final concentration of~~ 0.~~36g~~.mL-1. The resulting suspension was stirred on ice for 2 hours before further centrifugation (30 100 g, 20 minutes, 1º C). A small sample of the supernatant was retained for SDS-PAGE, but the remainder was discarded and the pellet was resuspended in a minimal volume (~1 mL) of Buffer C (50 mM imidazole-HCl, pH 6.8, 1 mM EDTA, 1 mM DTT). The ~~solution~~ was dialysed against two changes of 1 litre ~~of Buffer C~~.	+	The volume of the supernatant was measured. The NaCl concentration was adjusted to 1M and solid ammonium sulfate to a final concentration of 0.078 g.mL<sup>-1</sup> slowly added. The resulting suspension was stirred on ice for 1 hour and then centrifuged again (30 100 g, 20 minutes, 4ºC). The supernatant was retained and a sample was taken for SDS-PAGE analysis. The volume of the supernatant was again measured and solid ammonium sulfate was slowly add a further 0.29 g.mL<sup>-1</sup>. The resulting suspension was stirred on ice for 2 hours before further centrifugation (30 100 g, 20 minutes, 1º C). A small sample of the supernatant was retained for SDS-PAGE, but the remainder was discarded and the pellet was resuspended in a minimal volume (~1-5 mL) of Buffer A (50 mM imidazole-HCl, pH 6.8, 1 mM EDTA, 1 mM DTT). The suspension was then transferred to 10-12 kDa cut off dialysis tubing and dialysed against two changes of Buffer A (at least 1 litre each for at least one hour).

	Purification		Purification

-	The dialysed solution was diluted to ~2 ~~mg.mL-1 this was~~ loaded onto a Pharmacia DEAE FastFlow (~~1mL or~~ 5mL) pre-packed column equilibrated in Buffer C. The column was washed with at least three column volumes of Buffer C and the Tus protein was eluted in a gradient of 0.00 to 1.50 M NaCl ~~over~~ 10~~-100mL~~ in Buffer C. The fractions containing Tus were combined and ~~were~~ loaded directly onto a ~~SO3 monolithic column (BIA separations) or a 5mL Phosphocellulose~~ column equilibrated in Buffer C. The column was washed with 5 column volumes of Buffer C and the bound protein was eluted by the application of a gradient from 0.00 to 1.50 M NaCl in Buffer C as before.	+	The dialysed solution was diluted in Buffer A to an OD280 of ~2 and loaded onto a Pharmacia DEAE FastFlow (5mL) pre-packed column equilibrated in Buffer A. The column was washed with at least three column volumes of Buffer A and the Tus protein was eluted in a gradient of 0.00 to 1.50 M NaCl in 10 column volumes in Buffer A ####TUS-LPETGG-His6 MAY NOT ELUTE WHERE WE EXPECT - MAY BE IN FLOW THROUGH####. The fractions containing Tus were combined and loaded directly onto a 5-15 mL Whatmann P11 phosphocellulose column equilibrated in Buffer A. The column was washed with 5 column volumes of Buffer A and the bound protein was eluted by the application of a gradient from 0.00 to 1.50 M NaCl in Buffer A as before.

-	The resultant protein was exchanged into storage buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 20% w/v glycerol, 1 mM DTT)by dialysis or ~~using an Amicon 15mL spin concentrator~~. Following exchange the protein was concentrated to ~~~20μM~~ using a spin concentrator. The protein concentration was estimated from the UV Absorbance spectrum of the protein solution, and the samples were ~~aliquotted~~ and snap-frozen in liquid nitrogen before moving to store at –80 ºC.	+	The resultant protein was exchanged into storage buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 20% w/v glycerol, 1 mM DTT) by dialysis (at least three times >100 volumes) or with a desalting column equilibrated in Storage buffer. Following exchange the protein was concentrated to ~10-100 μM using a spin concentrator or Amicon. The protein concentration was estimated from the UV Absorbance spectrum of the protein solution, and the samples were aliquoted and snap-frozen in liquid nitrogen before moving to store at –80 ºC.

Cameron Neylon: added page

2008-07-21T11:53:59Z

added page

New page

Cell Lysis

The stored frozen cells Rosetta 2 pLysS/pCM862 were thawed at 4 ºC and resuspended in 15 mL.g-1 of ice-cold lysis buffer (50 mM Imidazole, pH 6.8, 1 mM EDTA 10% w/v sucrose, 0.05 M NaCl, 2 mM DTT, 10 mM spermidine-3HCl). Lysozyme was added to a concentration of 0.2 mg.mL-1 and the suspension was stirred on ice for 30 minutes. The cells were then subjected to three freeze-thaw cycles, by direct addition of liquid nitrogen, whilst stirring until frozen and then fully defrosting at room temperature, whilst stirring. Cell debris was removed from the suspension by centrifugation (30 100 g, 10 minutes, 4 ºC).

The volume of the supernatant was measured. The NaCl concentration was adjusted to 1M and solid ammonium sulfate to a final concentration of 15% slowly added. The resulting suspension was stirred on ice for 1 hour and then centrifuged again (30 100 g, 20 minutes, 4ºC). The supernatant was retained and a sample was taken for SDS-PAGE analysis. The volume of the supernatant was again measured and solid ammonium sulfate was slowly added to a final concentration of 0.36g.mL-1. The resulting suspension was stirred on ice for 2 hours before further centrifugation (30 100 g, 20 minutes, 1º C). A small sample of the supernatant was retained for SDS-PAGE, but the remainder was discarded and the pellet was resuspended in a minimal volume (~1 mL) of Buffer C (50 mM imidazole-HCl, pH 6.8, 1 mM EDTA, 1 mM DTT). The solution was dialysed against two changes of 1 litre of Buffer C.

Purification

The dialysed solution was diluted to ~2 mg.mL-1 this was loaded onto a Pharmacia DEAE FastFlow (1mL or 5mL) pre-packed column equilibrated in Buffer C. The column was washed with at least three column volumes of Buffer C and the Tus protein was eluted in a gradient of 0.00 to 1.50 M NaCl over 10-100mL in Buffer C. The fractions containing Tus were combined and were loaded directly onto a SO3 monolithic column (BIA separations) or a 5mL Phosphocellulose column equilibrated in Buffer C. The column was washed with 5 column volumes of Buffer C and the bound protein was eluted by the application of a gradient from 0.00 to 1.50 M NaCl in Buffer C as before.

The resultant protein was exchanged into storage buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 20% w/v glycerol, 1 mM DTT)by dialysis or using an Amicon 15mL spin concentrator. Following exchange the protein was concentrated to ~20μM using a spin concentrator. The protein concentration was estimated from the UV Absorbance spectrum of the protein solution, and the samples were aliquotted and snap-frozen in liquid nitrogen before moving to store at –80 ºC.