Purification of GST-HA-S6K - Revision history

Davebrid: updated dna amount in materials (was ok in protocol)

2012-09-20T19:59:23Z

updated dna amount in materials (was ok in protocol)

← Older revision		Revision as of 19:59, 20 September 2012
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	__NOTOC__		__NOTOC__
	==Materials==		==Materials==
	*GST-HA-S6K1 plasmid. Prepare by [[Cesium Chloride Preparation of DNA]]. Need ~~240~~ ug per 10 plates of cells		*GST-HA-S6K1 plasmid. Prepare by [[Cesium Chloride Preparation of DNA]]. Need 500 ug per 10 plates of cells
	*293A Cells.		*293A Cells.
	*Glutathione-Sepharose		*Glutathione-Sepharose

Davebrid at 18:26, 22 August 2012

2012-08-22T18:26:53Z

← Older revision		Revision as of 18:26, 22 August 2012
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	#Split 293A cells into 10-150 mm dishes in DMEM/FBS with '''no antibiotics'''.		#Split 293A cells into 10-150 mm dishes in DMEM/FBS with '''no antibiotics'''.
	#Transfect when cells are 90-95% confluent.		#Transfect when cells are 90-95% confluent.
	~~#Transfer cells to serum and P/S/G free media~~
	#Combine 500 ug DNA with 10 mL OptiMEM, and separately 700 uL Lipofectamine 2000 with 10 mL OptiMEM		#Combine 500 ug DNA with 10 mL OptiMEM, and separately 700 uL Lipofectamine 2000 with 10 mL OptiMEM
	#After 5 minutes, combine the DNA solution with the Lipofectamine solution.		#After 5 minutes, combine the DNA solution with the Lipofectamine solution.
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	#Treat cells for 30min with 100 nM Wortmannin to suppress endogenous S6K phosphorylation		#Treat cells for 30min with 100 nM Wortmannin to suppress endogenous S6K phosphorylation
	#Wash cells with D-PBS -/- twice (10 mL per plate)		#Wash cells with D-PBS -/- twice (10 mL per plate)
	#Add 0.5 mL [[HNTG Buffer]] ~~per~~ plate and scrape		#Add 0.5 mL [[HNTG Buffer]] to each plate and scrape
	#Collect scraped cells and incubate end over end in eppendorf tube for 30 min		#Collect scraped cells and incubate end over end in eppendorf tube for 30 min
	#Centrifuge in eppendorf centrifuge for 10 min at 14 000 RPM		#Centrifuge in eppendorf centrifuge for 10 min at 14 000 RPM

Davebrid: updated lipofectamine conditions

2012-08-22T18:18:52Z

updated lipofectamine conditions

← Older revision		Revision as of 18:18, 22 August 2012
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	==Protocol==		==Protocol==
	===Transfection of Cells===		===Transfection of Cells===
	#Split cells into 10 ~~new~~ dishes in DMEM/FBS with '''no antibiotics'''.		#Split 293A cells into 10-150 mm dishes in DMEM/FBS with '''no antibiotics'''.
	#~~Transefect with~~ 90-95% confluent		#Transfect when cells are 90-95% confluent.
	#Combine ~~240~~ ug DNA with ~~1.5~~ mL OptiMEM, and ~~600~~ uL Lipofectamine 2000 with ~~1.5~~ mL OptiMEM		#Transfer cells to serum and P/S/G free media
			#Combine 500 ug DNA with 10 mL OptiMEM, and separately 700 uL Lipofectamine 2000 with 10 mL OptiMEM
	#After 5 minutes, combine the DNA solution with the Lipofectamine solution.		#After 5 minutes, combine the DNA solution with the Lipofectamine solution.
	#Wait 20 min, then add 3 mL to each plate of cells		#Wait 20 min, then add ~2 mL to each plate of cells.
	#After ~4h refeed with normal media (~~containing~~ antibiotics)		#After ~4h refeed with normal media (can now contain antibiotics) and incubate overnight.

	===Purification of GST-S6K1===		===Purification of GST-S6K1===
			#Treat cells for 30min with 100 nM Wortmannin to suppress endogenous S6K phosphorylation
	#Wash cells with D-PBS -/- twice (10 mL per plate)		#Wash cells with D-PBS -/- twice (10 mL per plate)
	#Add 0.5 mL [[HNTG Buffer]] per plate and scrape		#Add 0.5 mL [[HNTG Buffer]] per plate and scrape

Davebrid at 17:13, 4 November 2009

2009-11-04T17:13:18Z

← Older revision		Revision as of 17:13, 4 November 2009
Line 1:		Line 1:
			__NOTOC__
	==Materials==		==Materials==
	*GST-HA-S6K1 plasmid. Prepare by [[Cesium Chloride Preparation of DNA]]. Need 240 ug per 10 plates of cells		*GST-HA-S6K1 plasmid. Prepare by [[Cesium Chloride Preparation of DNA]]. Need 240 ug per 10 plates of cells

Davebrid at 15:32, 4 November 2009

2009-11-04T15:32:51Z

← Older revision		Revision as of 15:32, 4 November 2009
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	#Collect supernatant (save a lysate sample) into a 15 mL Falcon tube		#Collect supernatant (save a lysate sample) into a 15 mL Falcon tube
	#Add 0.5 mL Glutatione-sepharose beads and incubate end over end for 1-2h.		#Add 0.5 mL Glutatione-sepharose beads and incubate end over end for 1-2h.

			[[Category: Protein Purification]]
			[[Category: mTOR]]
			[[Category: Kinase Assay]]

Davebrid: initial page

2009-11-04T15:32:12Z

initial page

New page

==Materials==
*GST-HA-S6K1 plasmid. Prepare by [[Cesium Chloride Preparation of DNA]]. Need 240 ug per 10 plates of cells
*293A Cells.
*Glutathione-Sepharose
*[[HNTG Buffer]]
*[[TORC1 Kinase Buffer]]
*Lipofectamine 2000
*OptiMEM

==Protocol==
===Transfection of Cells===
#Split cells into 10 new dishes in DMEM/FBS with '''no antibiotics'''.
#Transefect with 90-95% confluent
#Combine 240 ug DNA with 1.5 mL OptiMEM, and 600 uL Lipofectamine 2000 with 1.5 mL OptiMEM
#After 5 minutes, combine the DNA solution with the Lipofectamine solution.
#Wait 20 min, then add 3 mL to each plate of cells
#After ~4h refeed with normal media (containing antibiotics)

===Purification of GST-S6K1===
#Wash cells with D-PBS -/- twice (10 mL per plate)
#Add 0.5 mL [[HNTG Buffer]] per plate and scrape
#Collect scraped cells and incubate end over end in eppendorf tube for 30 min
#Centrifuge in eppendorf centrifuge for 10 min at 14 000 RPM
#Collect supernatant (save a lysate sample) into a 15 mL Falcon tube
#Add 0.5 mL Glutatione-sepharose beads and incubate end over end for 1-2h.