Silver: Lysate for Western - Revision history

Reshma P. Shetty at 18:12, 9 May 2007

Reshma P. Shetty — Wed, 09 May 2007 18:12:30 GMT

←Older revision		Revision as of 18:12, 9 May 2007
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	#Spin down at maximum speed in tabletop centrifuge for 10 min. (in cold room)		#Spin down at maximum speed in tabletop centrifuge for 10 min. (in cold room)
	#Remove supernatant (lysate) to another tube.		#Remove supernatant (lysate) to another tube.
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		+	[[Category:Protocol]]
		+	[[Category:In vitro]]
		+	[[Category:Yeast]]
		+	[[Category:Protein]]

JulianEskin at 20:57, 21 September 2005

JulianEskin — Wed, 21 Sep 2005 20:57:05 GMT

←Older revision		Revision as of 20:57, 21 September 2005
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	Back to [[Protocols\|OpenWetWare protocols]]		Back to [[Protocols\|OpenWetWare protocols]]
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	Back to [[Silver: Protocols\|Silver Lab protocols]]		Back to [[Silver: Protocols\|Silver Lab protocols]]
		+
	Back to [[Silver Lab]]		Back to [[Silver Lab]]

JulianEskin at 20:56, 21 September 2005

JulianEskin — Wed, 21 Sep 2005 20:56:39 GMT

←Older revision		Revision as of 20:56, 21 September 2005
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		+	Back to [[Protocols\|OpenWetWare protocols]]
		+	Back to [[Silver: Protocols\|Silver Lab protocols]]
		+	Back to [[Silver Lab]]
		+
		+
	#Grow cells		#Grow cells
	#Harvest		#Harvest

JulianEskin at 20:54, 21 September 2005

JulianEskin — Wed, 21 Sep 2005 20:54:17 GMT

New page

#Grow cells
#Harvest
#*Spin down
#*Wash with PBS
#*Spin down enough cells to collect a 50-100 µL cell pellet in a FastPrep tube
#Prepare lysis buffer (beforehand) and chill on ice
#*PBSMT:
#**2.5 mM MgCl<sub>2</sub>
#**3 mM KCl
#**0.5% Triton X-100
#**in PBS
#*Add protease inhibitors
#**PMSF: 1 mL per 100 mL buffer
#***Use fresh PMSF: dissolve 0.035g per 1mL 100%EtOH
#**PLAAC: 100 µL per 100 mL buffer
#**0.5 M Benzamidine: 260 µL per 100 mL buffer
#Add 50-100 µL of cold lysis buffer to cell pellet and vortex
#Add 1-2 scoops glass beads (bead level should be just below liquid level)
#Mix everything together by vortex
#Lyse in FastPrep bead beater
#*6.5 m/s
#*40 second lysis
#Add 500 µL lysis buffer (keep everything on ice!)
#Spin down at maximum speed in tabletop centrifuge for 10 min. (in cold room)
#Remove supernatant (lysate) to another tube.