3T3-L1 Adipocyte Fractionation - Revision history

Buchnerd: /* Fractionation */

2011-01-18T15:52:29Z

Fractionation

← Older revision		Revision as of 15:52, 18 January 2011
Line 15:		Line 15:
	#Centrifuge supernatant 75 min at 90 000 RPM in TLA 100.3 at 4C. Pellet is low density microsomes (LDM), supernatant is cytosol save a sample.		#Centrifuge supernatant 75 min at 90 000 RPM in TLA 100.3 at 4C. Pellet is low density microsomes (LDM), supernatant is cytosol save a sample.
	#To load equal volumes on a gel, load equal volumes of fractions. If doing optiprep, load twice as much volume of PNS and cytosol as MN/PM/HDM/LDM.		#To load equal volumes on a gel, load equal volumes of fractions. If doing optiprep, load twice as much volume of PNS and cytosol as MN/PM/HDM/LDM.
			#PM purification: Dissolve PM pellet in 1 mL HES, dounce 10 times, then load to 2 mL of 40% sucrose-cusion. Spin with SW41 rotor 42.1K (100,000g) for 1 hour. Discard top 1 mL (fat) and harvest 0.8 mL of PM containing fraction. Add 2.6 mL of HES in the fraction to dilute sucrose. Spin at 35,000 rpm for 20 min to pellet down PM.

	==Optiprep Gradient==		==Optiprep Gradient==

Davebrid: modified volumes to reflect whether using optiprep or not.

2010-04-28T14:05:46Z

modified volumes to reflect whether using optiprep or not.

Davebrid at 18:55, 10 September 2009

2009-09-10T18:55:52Z

← Older revision		Revision as of 18:55, 10 September 2009
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			__NOTOC__
	==Materials==		==Materials==
	*PBS		*PBS

Davebrid: /* References */

2009-09-10T18:46:21Z

References

← Older revision		Revision as of 18:46, 10 September 2009
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	==References==		==References==
	~~<pubmed>~~17765682~~</pubmed>~~		PMID 17765682

Davebrid: /* Optiprep Gradient */

2009-05-04T12:40:13Z

Optiprep Gradient

← Older revision		Revision as of 12:40, 4 May 2009
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	#Centrifuge 4h at 62 000 RPM in NVT90 at 4C.		#Centrifuge 4h at 62 000 RPM in NVT90 at 4C.
	#Cut off top of tube and gently remove 350 uL fractions to fresh tubes on ice.		#Cut off top of tube and gently remove 350 uL fractions to fresh tubes on ice.
	#Store samples at -80. Make up SDS samples and ~~<bold>~~do not boil~~</bold>~~		#Store samples at -80. Make up SDS samples and '''do not boil'''


	==References==		==References==
	<pubmed>17765682</pubmed>		<pubmed>17765682</pubmed>

Davebrid at 12:38, 4 May 2009

2009-05-04T12:38:08Z

← Older revision		Revision as of 12:38, 4 May 2009
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	*Lysis Buffer (25 mM HEPES, 250 mM Sucrose, PI tablet)		*Lysis Buffer (25 mM HEPES, 250 mM Sucrose, PI tablet)
	*Optiprep		*Optiprep
			*Cool centrifuges and rotors for JA17/JA25.5, TLA100.3 and NVT90 to 4C

	==Fractionation==		==Fractionation==
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	#Scrape 150 mm dish into 4 mL of Lysis Buffer		#Scrape 150 mm dish into 4 mL of Lysis Buffer
	#Homogenize in Dounce Homogenizer 20X on ice		#Homogenize in Dounce Homogenizer 20X on ice
	#Centrifuge 5 min at 3000g and collect post-nuclear supernatant (PNS). If desired resuspend pellet in 2 mL as mitochondria/nuclear fraction (MN)		#Centrifuge 5 min at 3000g in JA17 or JA25.5 and collect post-nuclear supernatant (PNS). If desired resuspend pellet in 2 mL as mitochondria/nuclear fraction (MN)
	#Centrifuge supernatant 15 min at 17 200g at 4C. Resuspend pellet in 2 mL as plasma membrane (PM). Save sample.		#Centrifuge supernatant 15 min at 17 200g at 4C in JA17 or JA25.5 with brake off. Resuspend pellet in 2 mL as plasma membrane (PM). Save sample.
	#Centrifuge supernatant 30 min at 48 000g at 4C. Resuspend pellet in 2 mL as high density microsomes (HDM). Save sample		#Centrifuge supernatant 30 min at 48 000g at 4C in JA17 or JA25.5 with brake off. Resuspend pellet in 2 mL as high density microsomes (HDM). Save sample
	#Centrifuge supernatant 75 min at 90 000 RPM in TLA 100.3 at 4C. Pellet is low density microsomes (LDM), supernatant is cytosol save a sample.		#Centrifuge supernatant 75 min at 90 000 RPM in TLA 100.3 at 4C. Pellet is low density microsomes (LDM), supernatant is cytosol save a sample.
	#To load equal volumes on a gel, load twice as much volume of PNS and cytosol as MN/PM/HDM/LDM.		#To load equal volumes on a gel, load twice as much volume of PNS and cytosol as MN/PM/HDM/LDM.

Davebrid: Created page with '==Materials== PBS Lysis Buffer (25 mM HEPES, 250 mM Sucrose, PI tablet) *Optiprep ==Fractionation== #Wash cells twice with ice cold PBS -/- #Scrape 150 mm dish into 4 mL of Ly...'

2009-05-04T12:35:55Z

Created page with '==Materials== *PBS *Lysis Buffer (25 mM HEPES, 250 mM Sucrose, PI tablet) *Optiprep ==Fractionation== #Wash cells twice with ice cold PBS -/- #Scrape 150 mm dish into 4 mL of Ly...'

New page

==Materials==
*PBS
*Lysis Buffer (25 mM HEPES, 250 mM Sucrose, PI tablet)
*Optiprep

==Fractionation==
#Wash cells twice with ice cold PBS -/-
#Scrape 150 mm dish into 4 mL of Lysis Buffer
#Homogenize in Dounce Homogenizer 20X on ice
#Centrifuge 5 min at 3000g and collect post-nuclear supernatant (PNS). If desired resuspend pellet in 2 mL as mitochondria/nuclear fraction (MN)
#Centrifuge supernatant 15 min at 17 200g at 4C. Resuspend pellet in 2 mL as plasma membrane (PM). Save sample.
#Centrifuge supernatant 30 min at 48 000g at 4C. Resuspend pellet in 2 mL as high density microsomes (HDM). Save sample
#Centrifuge supernatant 75 min at 90 000 RPM in TLA 100.3 at 4C. Pellet is low density microsomes (LDM), supernatant is cytosol save a sample.
#To load equal volumes on a gel, load twice as much volume of PNS and cytosol as MN/PM/HDM/LDM.

==Optiprep Gradient==
#Wash LDM fraction once with lysis buffer and resuspend in 2 mL of lysis buffer. Save sample as LDM
#Gently homogenize 10X in Dounce Homogenizer
#Mix optiprep to generate 18% gradient (1.5 mL optiprep + 3.5 mL LDM)
#Centrifuge 4h at 62 000 RPM in NVT90 at 4C.
#Cut off top of tube and gently remove 350 uL fractions to fresh tubes on ice.
#Store samples at -80. Make up SDS samples and <bold>do not boil</bold>

==References==
<pubmed>17765682</pubmed>

← Older revision		Revision as of 14:05, 28 April 2010
Line 11:		Line 11:
	#Homogenize in Dounce Homogenizer 20X on ice		#Homogenize in Dounce Homogenizer 20X on ice
	#Centrifuge 5 min at 3000g in JA17 or JA25.5 and collect post-nuclear supernatant (PNS). If desired resuspend pellet in 2 mL as mitochondria/nuclear fraction (MN)		#Centrifuge 5 min at 3000g in JA17 or JA25.5 and collect post-nuclear supernatant (PNS). If desired resuspend pellet in 2 mL as mitochondria/nuclear fraction (MN)
	#Centrifuge supernatant 15 min at 17 200g at 4C in JA17 or JA25.5 with brake off. Resuspend pellet in 2 mL as plasma membrane (PM). Save sample.		#Centrifuge supernatant 15 min at 17 200g at 4C in JA17 or JA25.5 with brake off. Resuspend pellet in 4 mL (2 mL if doing optiprep)as plasma membrane (PM). Save sample.
	#Centrifuge supernatant 30 min at 48 000g at 4C in JA17 or JA25.5 with brake off. Resuspend pellet in 2 mL as high density microsomes (HDM). Save sample		#Centrifuge supernatant 30 min at 48 000g at 4C in JA17 or JA25.5 with brake off. Resuspend pellet in 4 mL (2 mL if doing optiprep)as high density microsomes (HDM). Save sample
	#Centrifuge supernatant 75 min at 90 000 RPM in TLA 100.3 at 4C. Pellet is low density microsomes (LDM), supernatant is cytosol save a sample.		#Centrifuge supernatant 75 min at 90 000 RPM in TLA 100.3 at 4C. Pellet is low density microsomes (LDM), supernatant is cytosol save a sample.
	#To load equal volumes on a gel, load twice as much volume of PNS and cytosol as MN/PM/HDM/LDM.		#To load equal volumes on a gel, load equal volumes of fractions. If doing optiprep, load twice as much volume of PNS and cytosol as MN/PM/HDM/LDM.

	==Optiprep Gradient==		==Optiprep Gradient==
Line 22:		Line 22:
	#Centrifuge 4h at 62 000 RPM in NVT90 at 4C.		#Centrifuge 4h at 62 000 RPM in NVT90 at 4C.
	#Cut off top of tube and gently remove 350 uL fractions to fresh tubes on ice.		#Cut off top of tube and gently remove 350 uL fractions to fresh tubes on ice.
	#Store samples at -80. Make up SDS samples and '''do not boil'''		#Store samples at -80. Make up SDS samples and '''do not boil if probing for GLUT4'''

	==References==		==References==
	PMID 17765682		PMID 17765682