Griffin:Nuclear Extract Preparation - Revision history

Korey Griffin: /* Abstract */

Korey Griffin — Fri, 31 Oct 2008 04:33:33 GMT

Abstract

←Older revision		Revision as of 04:33, 31 October 2008
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	Nuclear extract preparation is a useful procedure to enrich nuclear proteins for various techniques including [http://en.wikipedia.org/wiki/Electrophoretic_mobility_shift_assay EMSA] and [http://en.wikipedia.org/wiki/Immunoprecipitation ChIP] studies. There are two separate protocols below;		Nuclear extract preparation is a useful procedure to enrich nuclear proteins for various techniques including [http://en.wikipedia.org/wiki/Electrophoretic_mobility_shift_assay EMSA] and [http://en.wikipedia.org/wiki/Immunoprecipitation ChIP] studies. There are two separate protocols below;

-	[[Image:nucleusmodel.jpg]The nucleus of a eukaryotic cell]	+	[[Image:nucleusmodel.jpg]]
		+	The nucleus of a eukaryotic cell

	==Hattori et al nuclear extract preparation==		==Hattori et al nuclear extract preparation==

Korey Griffin: /* Abstract */

Korey Griffin — Fri, 31 Oct 2008 04:33:14 GMT

Abstract

←Older revision		Revision as of 04:33, 31 October 2008
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	Nuclear extract preparation is a useful procedure to enrich nuclear proteins for various techniques including [http://en.wikipedia.org/wiki/Electrophoretic_mobility_shift_assay EMSA] and [http://en.wikipedia.org/wiki/Immunoprecipitation ChIP] studies. There are two separate protocols below;		Nuclear extract preparation is a useful procedure to enrich nuclear proteins for various techniques including [http://en.wikipedia.org/wiki/Electrophoretic_mobility_shift_assay EMSA] and [http://en.wikipedia.org/wiki/Immunoprecipitation ChIP] studies. There are two separate protocols below;

-	[[Image:nucleusmodel.jpg]]	+	[[Image:nucleusmodel.jpg]The nucleus of a eukaryotic cell]

	==Hattori et al nuclear extract preparation==		==Hattori et al nuclear extract preparation==

Korey Griffin: /* Abstract */

Korey Griffin — Fri, 31 Oct 2008 04:30:53 GMT

Abstract

←Older revision		Revision as of 04:30, 31 October 2008
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	Nuclear extract preparation is a useful procedure to enrich nuclear proteins for various techniques including [http://en.wikipedia.org/wiki/Electrophoretic_mobility_shift_assay EMSA] and [http://en.wikipedia.org/wiki/Immunoprecipitation ChIP] studies. There are two separate protocols below;		Nuclear extract preparation is a useful procedure to enrich nuclear proteins for various techniques including [http://en.wikipedia.org/wiki/Electrophoretic_mobility_shift_assay EMSA] and [http://en.wikipedia.org/wiki/Immunoprecipitation ChIP] studies. There are two separate protocols below;
		+
		+	[[Image:nucleusmodel.jpg]]

	==Hattori et al nuclear extract preparation==		==Hattori et al nuclear extract preparation==

Korey Griffin at 18:31, 30 October 2008

Korey Griffin — Thu, 30 Oct 2008 18:31:23 GMT

←Older revision		Revision as of 18:31, 30 October 2008
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	Nuclear extract preparation is a useful procedure to enrich nuclear proteins for various techniques including [http://en.wikipedia.org/wiki/Electrophoretic_mobility_shift_assay EMSA] and [http://en.wikipedia.org/wiki/Immunoprecipitation ChIP] studies. There are two separate protocols below;		Nuclear extract preparation is a useful procedure to enrich nuclear proteins for various techniques including [http://en.wikipedia.org/wiki/Electrophoretic_mobility_shift_assay EMSA] and [http://en.wikipedia.org/wiki/Immunoprecipitation ChIP] studies. There are two separate protocols below;

-	===Hattori et al nuclear extract preparation===	+	==Hattori et al nuclear extract preparation==

-	==Reagents I==	+	===Reagents I===
	'''I. Homogenization buffer:'''		'''I. Homogenization buffer:'''

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	10ml of ethanol		10ml of ethanol

-	==Procedure I==	+	===Procedure I===

	'''I. Homogenize cells or tissue'''		'''I. Homogenize cells or tissue'''
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	3. Measure protein and aliquot supernatant (100-500mg/tube) and store it at -70C		3. Measure protein and aliquot supernatant (100-500mg/tube) and store it at -70C

-	==References I==	+	===References I===
	<biblio>		<biblio>
	#Paper1 pmid=2264931		#Paper1 pmid=2264931
	</biblio>		</biblio>

		+	==Dignam et al nuclear extract preparation==

-	=~~==Dignam et al nuclear extract preparation===~~	+	===Reagents II===
-		+
-	==Reagents II==	+
	Buffers used for extract preparation are designated as follows:		Buffers used for extract preparation are designated as follows:

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	'''Buffer D:''' 20 mM HEPES (pH 7.9), 20% (v/v) glycerol, 0.1 M KCL, 0.2 mM EDTA, 0.5 mM PMSF, and 0.5 mM DTT. DTT and PMSF were added fresh to the buffers just before use.		'''Buffer D:''' 20 mM HEPES (pH 7.9), 20% (v/v) glycerol, 0.1 M KCL, 0.2 mM EDTA, 0.5 mM PMSF, and 0.5 mM DTT. DTT and PMSF were added fresh to the buffers just before use.

-	==Procedure II==	+	===Procedure II===

	Hela cells were grown in spinner flasks at 37oC in Joklik’s MEM containing 5% calf serum. They were frown to 4 to 6 x 105 cells per ml prior to harvesting for extract preparation.		Hela cells were grown in spinner flasks at 37oC in Joklik’s MEM containing 5% calf serum. They were frown to 4 to 6 x 105 cells per ml prior to harvesting for extract preparation.
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	The protein concentration was usually 6-8 mg/ml and 15-20 mg of protein were obtained from 109 cells.		The protein concentration was usually 6-8 mg/ml and 15-20 mg of protein were obtained from 109 cells.

-	==References II==	+	===References II===
	<biblio>		<biblio>
	#Paper1 pmid=6828386		#Paper1 pmid=6828386

Korey Griffin at 18:30, 30 October 2008

Korey Griffin — Thu, 30 Oct 2008 18:30:16 GMT

←Older revision		Revision as of 18:30, 30 October 2008
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	Nuclear extract preparation is a useful procedure to enrich nuclear proteins for various techniques including [http://en.wikipedia.org/wiki/Electrophoretic_mobility_shift_assay EMSA] and [http://en.wikipedia.org/wiki/Immunoprecipitation ChIP] studies. There are two separate protocols below;		Nuclear extract preparation is a useful procedure to enrich nuclear proteins for various techniques including [http://en.wikipedia.org/wiki/Electrophoretic_mobility_shift_assay EMSA] and [http://en.wikipedia.org/wiki/Immunoprecipitation ChIP] studies. There are two separate protocols below;
-
-	~~[[Image:nucleusmodel.jpg]]~~

	===Hattori et al nuclear extract preparation===		===Hattori et al nuclear extract preparation===

Korey Griffin at 18:29, 30 October 2008

Korey Griffin — Thu, 30 Oct 2008 18:29:16 GMT

←Older revision		Revision as of 18:29, 30 October 2008
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	==Curators==		==Curators==
-	''Anyone should feel free to add themselves as a curator for this consensus protocol. You do not need to be a curator in order to contribute. The OpenWetWare community is currently [[OpenWetWare:Information management/Protocol curators\|discussing the idea of protocol curators]]. Please contribute.''

	[http://www.openwetware.org/wiki/User:Korey_Griffin Korey Griffin]		[http://www.openwetware.org/wiki/User:Korey_Griffin Korey Griffin]
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	Nuclear extract preparation is a useful procedure to enrich nuclear proteins for various techniques including [http://en.wikipedia.org/wiki/Electrophoretic_mobility_shift_assay EMSA] and [http://en.wikipedia.org/wiki/Immunoprecipitation ChIP] studies. There are two separate protocols below;		Nuclear extract preparation is a useful procedure to enrich nuclear proteins for various techniques including [http://en.wikipedia.org/wiki/Electrophoretic_mobility_shift_assay EMSA] and [http://en.wikipedia.org/wiki/Immunoprecipitation ChIP] studies. There are two separate protocols below;
		+
		+	[[Image:nucleusmodel.jpg]]

	===Hattori et al nuclear extract preparation===		===Hattori et al nuclear extract preparation===

Korey Griffin: /* Reagents I */

Korey Griffin — Mon, 22 Sep 2008 17:05:18 GMT

Reagents I

←Older revision		Revision as of 17:05, 22 September 2008
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	Nuclear extract preparation is a useful procedure to enrich nuclear proteins for various techniques including [http://en.wikipedia.org/wiki/Electrophoretic_mobility_shift_assay EMSA] and [http://en.wikipedia.org/wiki/Immunoprecipitation ChIP] studies. There are two separate protocols below;		Nuclear extract preparation is a useful procedure to enrich nuclear proteins for various techniques including [http://en.wikipedia.org/wiki/Electrophoretic_mobility_shift_assay EMSA] and [http://en.wikipedia.org/wiki/Immunoprecipitation ChIP] studies. There are two separate protocols below;
		+
		+	===Hattori et al nuclear extract preparation===

	==Reagents I==		==Reagents I==

Korey Griffin: /* References I */

Korey Griffin — Mon, 22 Sep 2008 17:04:30 GMT

References I

←Older revision		Revision as of 17:04, 22 September 2008
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	</biblio>		</biblio>

-	~~----~~

	===Dignam et al nuclear extract preparation===		===Dignam et al nuclear extract preparation===

Korey Griffin: /* Reagents II */

Korey Griffin — Mon, 22 Sep 2008 17:04:07 GMT

Reagents II

←Older revision		Revision as of 17:04, 22 September 2008
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	----		----
		+
		+	===Dignam et al nuclear extract preparation===

	==Reagents II==		==Reagents II==

Korey Griffin: /* Reagents II */

Korey Griffin — Mon, 22 Sep 2008 17:03:15 GMT

Reagents II

←Older revision		Revision as of 17:03, 22 September 2008
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	----		----

-	===Reagents II===	+	==Reagents II==
	Buffers used for extract preparation are designated as follows:		Buffers used for extract preparation are designated as follows: