Preparing Cell Lysates - Revision history

Davebrid: /* RIPA Buffer (for 10mL lysis buffer) */ added link to orthovanadate protocol

2016-01-27T20:57:36Z

RIPA Buffer (for 10mL lysis buffer): added link to orthovanadate protocol

← Older revision		Revision as of 20:57, 27 January 2016
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	\|EDTA \|\| 1mM \|\| 20uL \|\| 0.5M		\|EDTA \|\| 1mM \|\| 20uL \|\| 0.5M
	\|-		\|-
	\|NaVO3 \|\| 100uM \|\| 10uL \|\| 100mM		\|NaVO3 (see [[Preparation_of_Orthovanadate_Stocks \| preparation ]]) \|\| 100uM \|\| 10uL \|\| 100mM
	\|-		\|-
	\|NaF \|\| 5mM \|\| 100uL \|\| 0.5M		\|NaF \|\| 5mM \|\| 100uL \|\| 0.5M

2009-11-03T16:19:27Z

Basic Protocol

← Older revision		Revision as of 16:19, 3 November 2009
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	# Stimulate cells if necessary (i.e. insulin treatment for 10 min)		# Stimulate cells if necessary (i.e. insulin treatment for 10 min)
	# Wash cells 2x1mL (1mL per well for 12 well plates, 2mL per well for 6 well plates) with ice cold PBS -/- and aspirate		# Wash cells 2x1mL (1mL per well for 12 well plates, 2mL per well for 6 well plates) with ice cold PBS -/- and aspirate
	# Add 200uL [[Buffer:RIPA]] buffer and scrape cells		# Add 200uL Buffer (RIPA buffer) and scrape cells
	# Pipet into cold eppendorf tubes		# Pipet into cold eppendorf tubes
	# rotate end over end for 30 minutes at 4oC to lyse		# rotate end over end for 30 minutes at 4oC to lyse

2009-11-03T16:18:10Z

RIPA Buffer (for 10mL lysis buffer)

← Older revision		Revision as of 16:18, 3 November 2009
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	\|NaPPi \|\| 25 mM \|\| 1 mL \|\| 250mM		\|NaPPi \|\| 25 mM \|\| 1 mL \|\| 250mM
	\|-		\|-
	\|- Protease Inhibitors !! 1 mini tab		\|Protease Inhibitors \|\| \|\| 1 mini tab \|\|
	\|}		\|}
	~~\|NaPPi \|\| 25 mM \|\| 1 mL \|\| 250mM~~

	==Basic Protocol==		==Basic Protocol==

2009-11-03T16:15:59Z

RIPA Buffer (for 10mL lysis buffer)

← Older revision		Revision as of 16:15, 3 November 2009
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	\|- Protease Inhibitors !! 1 mini tab		\|- Protease Inhibitors !! 1 mini tab
	\|}		\|}
			\|NaPPi \|\| 25 mM \|\| 1 mL \|\| 250mM

	==Basic Protocol==		==Basic Protocol==

2009-11-03T16:14:17Z

Basic Protocol

← Older revision		Revision as of 16:14, 3 November 2009
Line 25:		Line 25:

	==Basic Protocol==		==Basic Protocol==
	# Stimulate cells if necessary		# Stimulate cells if necessary (i.e. insulin treatment for 10 min)
	# Wash cells 2x1mL with ice cold PBS -/- and aspirate		# Wash cells 2x1mL (1mL per well for 12 well plates, 2mL per well for 6 well plates) with ice cold PBS -/- and aspirate
	# Add 200uL [[Buffer:RIPA]] buffer and scrape cells		# Add 200uL [[Buffer:RIPA]] buffer and scrape cells
	# Pipet into cold eppendorf tubes		# Pipet into cold eppendorf tubes
	# rotate end over end for 30 minutes at 4C to lyse		# rotate end over end for 30 minutes at 4oC to lyse
	# Centrifuge 10 min at 13,000 RPM to clarify		# Centrifuge 10 min at 13,000 RPM to clarify
	# Transfer 150uL of lysate to fresh tube and add 150uL 2XSDS		# Transfer 150uL of lysate to fresh tube and do Bradford assay before add 150uL 2XSDS
	# Load gel		# Load gel

2009-11-03T16:10:41Z

RIPA Buffer (for 10mL lysis buffer)

← Older revision		Revision as of 16:10, 3 November 2009
Line 5:		Line 5:
	! \|\| Final Concentration \|\| per 10 mL \|\| Stock		! \|\| Final Concentration \|\| per 10 mL \|\| Stock
	\|-		\|-
	\|Tris pH7.4 \|\| 50mM \|\| 500uL \|\| 1M		\|Tris pH7.4 \|\| 50mM \|\| 500uL \|\| 1M (pH 8.0@25oC)
	\|-		\|-
	\|Na Deoxycholate \|\| 0.25% \|\| 250uL \|\| 10%		\|Na Deoxycholate \|\| 0.25% \|\| 250uL \|\| 10%
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	\|EDTA \|\| 1mM \|\| 20uL \|\| 0.5M		\|EDTA \|\| 1mM \|\| 20uL \|\| 0.5M
	\|-		\|-
	\|NaVO3 \|\| ~~1mM~~ \|\| ~~100uL~~ \|\| 1M		\|NaVO3 \|\| 100uM \|\| 10uL \|\| 100mM
	\|-		\|-
	\|NaF \|\| 5mM \|\| 100uL \|\| ~~250mM~~		\|NaF \|\| 5mM \|\| 100uL \|\| 0.5M
	\|-		\|-
	\|NaPPi \|\| 25 mM \|\| 1 mL \|\| ~~500mM~~		\|NaPPi \|\| 25 mM \|\| 1 mL \|\| 250mM
	\|-		\|-
	\|- Protease Inhibitors !! 1 tab		\|- Protease Inhibitors !! 1 mini tab
	\|}		\|}

2009-07-15T15:15:09Z

← Older revision		Revision as of 15:15, 15 July 2009
Line 27:		Line 27:
	# Stimulate cells if necessary		# Stimulate cells if necessary
	# Wash cells 2x1mL with ice cold PBS -/- and aspirate		# Wash cells 2x1mL with ice cold PBS -/- and aspirate
	# Add 200uL [[Buffer~~/RIPA\|~~RIPA]] buffer and scrape cells		# Add 200uL [[Buffer:RIPA]] buffer and scrape cells
	# Pipet into cold eppendorf tubes		# Pipet into cold eppendorf tubes
	# rotate end over end for 30 minutes at 4C to lyse		# rotate end over end for 30 minutes at 4C to lyse

2009-05-27T00:32:28Z

Basic Protocol

← Older revision		Revision as of 00:32, 27 May 2009
Line 27:		Line 27:
	# Stimulate cells if necessary		# Stimulate cells if necessary
	# Wash cells 2x1mL with ice cold PBS -/- and aspirate		# Wash cells 2x1mL with ice cold PBS -/- and aspirate
	# Add 200uL RIPA buffer and scrape cells		# Add 200uL [[Buffer/RIPA\|RIPA]] buffer and scrape cells
	# Pipet into cold eppendorf tubes		# Pipet into cold eppendorf tubes
	# rotate end over end for 30 minutes at 4C to lyse		# rotate end over end for 30 minutes at 4C to lyse

2009-05-26T21:25:37Z

edited phosphatase inhibitors

← Older revision		Revision as of 21:25, 26 May 2009
Line 2:		Line 2:

	===RIPA Buffer (for 10mL lysis buffer)===		===RIPA Buffer (for 10mL lysis buffer)===
	{border = "1" ~~cellspacing = "0" cellpadding = "5"~~		{\| border="1"
	\|-Tris pH7.4 !! 50mM !! 500uL		! \|\| Final Concentration \|\| per 10 mL \|\| Stock
	\|-Na Deoxycholate !! 0.25% !! 250uL		\|-
	\|-NP-40 !! 1% !! 1mL		\|Tris pH7.4 \|\| 50mM \|\| 500uL \|\| 1M
	\|-NaCl !! 150mM !! 375uL		\|-
	\|-EDTA !! 1mM !! 20uL		\|Na Deoxycholate \|\| 0.25% \|\| 250uL \|\| 10%
	\|-NaVO3 !! 1mM !! 100uL		\|-
	\|-NaF ~~!! 1mM !! 20uL~~		\| NP-40 \|\| 1% \|\| 1mL \|\| 10%
	\|-Protease Inhibitors !! 1 tab		\|-
	}		\|NaCl \|\| 150mM \|\| 375uL \|\| 4M
			\|-
			\|EDTA \|\| 1mM \|\| 20uL \|\| 0.5M
			\|-
			\|NaVO3 \|\| 1mM \|\| 100uL \|\| 1M
			\|-
			\|NaF \|\| 5mM \|\| 100uL \|\| 250mM
			\|-
			\|NaPPi \|\| 25 mM \|\| 1 mL \|\| 500mM
			\|-
			\|- Protease Inhibitors !! 1 tab
			\|}

	==Basic Protocol==		==Basic Protocol==

2009-05-26T21:15:49Z

RIPA Buffer (for 10mL lysis buffer)