Drummond:Lysis - Revision history

Dadrummond at 19:36, 18 March 2009

2009-03-18T19:36:27Z

←Older revision		Revision as of 19:36, 18 March 2009
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		+	==Overview==
		+	More thoughts: [[Drummond:Solubility\|Extracting soluble and insoluble protein fractions]]
		+
	==Mechanical lysis==		==Mechanical lysis==
	Most popular method is bead-beating. Advantages: fast, cheap, widely used. Disadvantages: messy, incomplete lysis, evidence of protein denaturation?		Most popular method is bead-beating. Advantages: fast, cheap, widely used. Disadvantages: messy, incomplete lysis, evidence of protein denaturation?

Dadrummond: more sections

2009-02-08T22:30:36Z

more sections

←Older revision		Revision as of 22:30, 8 February 2009
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		+	==Mechanical lysis==
		+	Most popular method is bead-beating. Advantages: fast, cheap, widely used. Disadvantages: messy, incomplete lysis, evidence of protein denaturation?
		+
		+	==Enzymatic lysis==
		+	Zymolyase works cleanly and with high efficiency. However, it requires a long incubation period (30-45min) during which we see evidence of protein degradation. Unclear if this is a stress response or something else.
		+
	==Chemical lysis==		==Chemical lysis==
	Chemical lysis is an attractive route for extracting yeast proteins, because enzymatic treatments leave extraneous proteins in the sample and mechanical lysis, e.g. with beads, is a bit of a pain. While several companies sell chemical lysis reagents, such as Novagen's [http://www.emdbiosciences.com/html/NVG/yeastbuster.htm YeastBuster] and Pierce's [http://www.piercenet.com/products/browse.cfm?fldID=06010430 Y-Per], the formulations are proprietary and it's not clear what the tradeoffs might be.		Chemical lysis is an attractive route for extracting yeast proteins, because enzymatic treatments leave extraneous proteins in the sample and mechanical lysis, e.g. with beads, is a bit of a pain. While several companies sell chemical lysis reagents, such as Novagen's [http://www.emdbiosciences.com/html/NVG/yeastbuster.htm YeastBuster] and Pierce's [http://www.piercenet.com/products/browse.cfm?fldID=06010430 Y-Per], the formulations are proprietary and it's not clear what the tradeoffs might be.

Dadrummond: links

2007-07-07T19:43:05Z

links

←Older revision		Revision as of 19:43, 7 July 2007
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-
	==Chemical lysis==		==Chemical lysis==
		+	Chemical lysis is an attractive route for extracting yeast proteins, because enzymatic treatments leave extraneous proteins in the sample and mechanical lysis, e.g. with beads, is a bit of a pain. While several companies sell chemical lysis reagents, such as Novagen's [http://www.emdbiosciences.com/html/NVG/yeastbuster.htm YeastBuster] and Pierce's [http://www.piercenet.com/products/browse.cfm?fldID=06010430 Y-Per], the formulations are proprietary and it's not clear what the tradeoffs might be.
		+
	The major active ingredient in Y-Per lysis reagent is apparently 10 mg/mL SB3-14 (myristyl sulfobetaine)<cite>ZannaJChr07</cite>, also known as		The major active ingredient in Y-Per lysis reagent is apparently 10 mg/mL SB3-14 (myristyl sulfobetaine)<cite>ZannaJChr07</cite>, also known as
	3-(N-tetradecyl-N,N-dimethyl-ammonio)-propane-1-sulfonate or SB14, available from Sigma as [http://www.sigmaaldrich.com/catalog/search/ProductDetail/FLUKA/40772 SB3-14]. Y-Per is a protein-free reagent and SB3-14 is the only detergent <cite>ZannaJChro07</cite>.		3-(N-tetradecyl-N,N-dimethyl-ammonio)-propane-1-sulfonate or SB14, available from Sigma as [http://www.sigmaaldrich.com/catalog/search/ProductDetail/FLUKA/40772 SB3-14]. Y-Per is a protein-free reagent and SB3-14 is the only detergent <cite>ZannaJChro07</cite>.

Dadrummond: rearranging

2007-07-07T19:37:41Z

rearranging

←Older revision		Revision as of 19:37, 7 July 2007
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		+
		+	==Chemical lysis==
		+	The major active ingredient in Y-Per lysis reagent is apparently 10 mg/mL SB3-14 (myristyl sulfobetaine)<cite>ZannaJChr07</cite>, also known as
		+	3-(N-tetradecyl-N,N-dimethyl-ammonio)-propane-1-sulfonate or SB14, available from Sigma as [http://www.sigmaaldrich.com/catalog/search/ProductDetail/FLUKA/40772 SB3-14]. Y-Per is a protein-free reagent and SB3-14 is the only detergent <cite>ZannaJChro07</cite>.
		+
		+	For assessing soluble versus insoluble fractions of proteins, chemical lysis of membranes may not be ideal. The problem is that if detergents liberate proteins from the membrane efficiently, then we can expect them to solubilize some insoluble proteins, and reduce signal. The appeal of mechanical lysis is that it is not expected to alter the solubility of proteins.
		+
		+	Idea: Mechanically disrupt, spin, and extract supernatant. Spin down to clarify. Draw off supernatant as soluble fraction and save pellet. Wash beads with 100 μL solubilization buffer, vortex, and draw off as much material as possible (liquid + pellet). Add to pellet remaining after soluble fraction removal. Vortex to resuspend and incubate 5 min at room temperature. Spin down to clarify. Draw off supernatant as insoluble fraction.
		+
		+	==Notes==
	SUME Buffer		SUME Buffer
	(1% SDS, 8M Urea, 10mM MOPS, pH 6.8, 10mM EDTA)		(1% SDS, 8M Urea, 10mM MOPS, pH 6.8, 10mM EDTA)
Line 11:		Line 21:

	from [http://www.fhcrc.org/science/labs/gottschling/yeast/yeastip.html?&&printfriendly=yes Yeast Protocols]		from [http://www.fhcrc.org/science/labs/gottschling/yeast/yeastip.html?&&printfriendly=yes Yeast Protocols]
-
-	~~==Chemical lysis==~~
-	~~The major active ingredient in Y-Per lysis reagent is apparently 10 mg/mL SB3-14 (myristyl sulfobetaine)<cite>ZannaJChr07</cite>, also known as~~
-	3-(N-tetradecyl-N,N-dimethyl-ammonio)-propane-1-sulfonate or SB14, available from Sigma as [http://www.sigmaaldrich.com/catalog/search/ProductDetail/FLUKA/40772 SB3-14]. Y-Per is a protein-free reagent and SB3-14 is the only detergent <cite>ZannaJChro07</cite>.
-
-	For assessing soluble versus insoluble fractions of proteins, chemical lysis of membranes may not be ideal. The problem is that if detergents liberate proteins from the membrane efficiently, then we can expect them to solubilize some insoluble proteins, and reduce signal. The appeal of mechanical lysis is that it is not expected to alter the solubility of proteins.
-
-	Idea: Mechanically disrupt, spin, and extract supernatant. Spin down to clarify. Draw off supernatant as soluble fraction and save pellet. Wash beads with 100 μL solubilization buffer, vortex, and draw off as much material as possible (liquid + pellet). Add to pellet remaining after soluble fraction removal. Vortex to resuspend and incubate 5 min at room temperature. Spin down to clarify. Draw off supernatant as insoluble fraction.

	==References==		==References==

Dadrummond: cheaper SB3-14

2007-07-07T18:48:32Z

cheaper SB3-14

←Older revision		Revision as of 18:48, 7 July 2007
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	==Chemical lysis==		==Chemical lysis==
	The major active ingredient in Y-Per lysis reagent is apparently 10 mg/mL SB3-14 (myristyl sulfobetaine)<cite>ZannaJChr07</cite>, also known as		The major active ingredient in Y-Per lysis reagent is apparently 10 mg/mL SB3-14 (myristyl sulfobetaine)<cite>ZannaJChr07</cite>, also known as
-	3-(N-tetradecyl-N,N-dimethyl-ammonio)-propane-1-sulfonate or SB14, available from Sigma as [http://www.sigmaaldrich.com/catalog/search/ProductDetail/~~SIGMA~~/~~T7763~~ SB3-14]. Y-Per is a protein-free reagent and SB3-14 is the only detergent <cite>ZannaJChro07</cite>.	+	3-(N-tetradecyl-N,N-dimethyl-ammonio)-propane-1-sulfonate or SB14, available from Sigma as [http://www.sigmaaldrich.com/catalog/search/ProductDetail/FLUKA/40772 SB3-14]. Y-Per is a protein-free reagent and SB3-14 is the only detergent <cite>ZannaJChro07</cite>.

	For assessing soluble versus insoluble fractions of proteins, chemical lysis of membranes may not be ideal. The problem is that if detergents liberate proteins from the membrane efficiently, then we can expect them to solubilize some insoluble proteins, and reduce signal. The appeal of mechanical lysis is that it is not expected to alter the solubility of proteins.		For assessing soluble versus insoluble fractions of proteins, chemical lysis of membranes may not be ideal. The problem is that if detergents liberate proteins from the membrane efficiently, then we can expect them to solubilize some insoluble proteins, and reduce signal. The appeal of mechanical lysis is that it is not expected to alter the solubility of proteins.

Dadrummond: Y-Per; thoughts on chemical lysis

2007-07-07T18:47:06Z

Y-Per; thoughts on chemical lysis

←Older revision		Revision as of 18:47, 7 July 2007
Line 13:		Line 13:

	==Chemical lysis==		==Chemical lysis==
-	~~Myristyl~~ sulfobetaine	+	The major active ingredient in Y-Per lysis reagent is apparently 10 mg/mL SB3-14 (myristyl sulfobetaine)<cite>ZannaJChr07</cite>, also known as
-	3-(N-tetradecyl-N,N-dimethyl-ammonio)-propane-1-sulfonate (SB14~~) <cite>ZannaJChr07</cite>~~, available from Sigma as [http://www.sigmaaldrich.com/catalog/search/ProductDetail/SIGMA/T7763 SB3-14].	+	3-(N-tetradecyl-N,N-dimethyl-ammonio)-propane-1-sulfonate or SB14, available from Sigma as [http://www.sigmaaldrich.com/catalog/search/ProductDetail/SIGMA/T7763 SB3-14]. Y-Per is a protein-free reagent and SB3-14 is the only detergent <cite>ZannaJChro07</cite>.
		+
		+	For assessing soluble versus insoluble fractions of proteins, chemical lysis of membranes may not be ideal. The problem is that if detergents liberate proteins from the membrane efficiently, then we can expect them to solubilize some insoluble proteins, and reduce signal. The appeal of mechanical lysis is that it is not expected to alter the solubility of proteins.
		+
		+	Idea: Mechanically disrupt, spin, and extract supernatant. Spin down to clarify. Draw off supernatant as soluble fraction and save pellet. Wash beads with 100 μL solubilization buffer, vortex, and draw off as much material as possible (liquid + pellet). Add to pellet remaining after soluble fraction removal. Vortex to resuspend and incubate 5 min at room temperature. Spin down to clarify. Draw off supernatant as insoluble fraction.

	==References==		==References==

Dadrummond: Zanna

2007-07-07T18:00:58Z

Zanna

←Older revision		Revision as of 18:00, 7 July 2007
Line 11:		Line 11:

	from [http://www.fhcrc.org/science/labs/gottschling/yeast/yeastip.html?&&printfriendly=yes Yeast Protocols]		from [http://www.fhcrc.org/science/labs/gottschling/yeast/yeastip.html?&&printfriendly=yes Yeast Protocols]
		+
		+	==Chemical lysis==
		+	Myristyl sulfobetaine
		+	3-(N-tetradecyl-N,N-dimethyl-ammonio)-propane-1-sulfonate (SB14) <cite>ZannaJChr07</cite>, available from Sigma as [http://www.sigmaaldrich.com/catalog/search/ProductDetail/SIGMA/T7763 SB3-14].
		+
		+	==References==
		+	<biblio>
		+	#ZannaJChr07 pmid=16978932
		+	</biblio>

Dadrummond: protocols page

2007-06-21T18:26:05Z

protocols page

←Older revision		Revision as of 18:26, 21 June 2007
Line 9:		Line 9:

	This is used for lysing yeast at pH6.8. For pH8 lyses, use the variation of SUME with Tris buffer known as SUTE.		This is used for lysing yeast at pH6.8. For pH8 lyses, use the variation of SUME with Tris buffer known as SUTE.
		+
		+	from [http://www.fhcrc.org/science/labs/gottschling/yeast/yeastip.html?&&printfriendly=yes Yeast Protocols]

Dadrummond: SUME buffer

2007-06-21T18:25:39Z

SUME buffer

New page

SUME Buffer
(1% SDS, 8M Urea, 10mM MOPS, pH 6.8, 10mM EDTA)
To make 100ml:

* 1.0ml 1M MOPS, pH6.8 stock solution
* 1.0g SDS, or 10ml 10% SDS stock solution
* 48.05g Urea
* 2.0ml 0.5M EDTA stock solution

This is used for lysing yeast at pH6.8. For pH8 lyses, use the variation of SUME with Tris buffer known as SUTE.