Bridges Lab Protocols - User contributions [en]

3T3-L1 Plasma Membrane Isolation

2011-01-21T21:44:49Z

Buchnerd:

#Wash cells 3x in cold HES (20 mM Hepes, pH 7.4; 1 mM EDTA, 255 mM Sucrose)
#Dry cells well and add 0.6 mL HES (w/Protease Inhibitor) per 150 mm dish
#Scrape cells and homogenize with dounce homogenizer for 20 strokes
#Centrifuge @ 3,000 rpm (800g) at 4 degrees for 10 minutes
#(Optional) Collect supernatant and spin @ 3,000 rpm at 4 degrees for 5 minutes
#Collect supernatant and spin in TLA 100.3 rotor at 19,000 rpm for 20 minutes at 4 degress (Tube = Beckman #349622)
#(Optional) Wash pellet in 1.0 mL HES and spin in TLA 100.3 rotor at 19,000 rpm for 20 minutes at 4 degress (Tube = Beckman #349622)
#(Optional) To further purify PM, resuspend in 0.6 mL HES and repeat steps #4 and #6
#Resuspend pellet in 1.0 mL HES and homogenize with dounce for 10 strokes
#Layer the resuspended pellet on top of 10 mL 40% sucrose/HES in a 13.5 mL ultracentrifuge tube (tube = Beckman #331372)
#Top off centrifuge tube with HES until nearly full
#Spin in ultracentrifuge with rotor SW41 (saltiel login = 5238) at 39,000 rpm at 4 degrees for 1 hour
#PM should appear as band at the interface of the HES and Sucrose fractions
#Remove HES buffer above PM fraction until close to PM fraction
#Collect ~ 0.8 mL PM containing fraction
#Add 2.6 mL of HES to dilute sucrose
#Spin in ultracentrifuge with TLA 100.3 rotor at 35,000 rpm for 20 minutes at 4 degress (Tube = Beckman #349622)
#Discard supernatent
#Pellet = purified plasma membrane

3T3-L1 Plasma Membrane Isolation

2011-01-21T21:41:59Z

Buchnerd:

#Wash cells 3x in cold HES (20 mM Hepes, pH 7.4; 1 mM EDTA, 255 mM Sucrose)
#Dry cells well and add 0.6 mL HES (w/Protease Inhibitor) per 150 mm dish
#Scrape cells and homogenize with dounce homogenizer for 20 strokes
#Centrifuge @ 3,000 rpm (800g) at 4 degrees for 10 minutes
#(Optional) Collect supernatant and spin @ 3,000 rpm at 4 degrees for 5 minutes
#Collect supernatant and spin in TLA 100.3 rotor at 19,000 rpm for 20 minutes at 4 degress (Tube = Beckman #349622)
#(Optional) Wash pellet in 1.0 mL HES and spin in TLA 100.3 rotor at 19,000 rpm for 20 minutes at 4 degress (Tube = Beckman #349622)
#(Optional) To further purify PM, repeat steps #4 and #6
#Resuspend pellet in 1.0 mL HES and homogenize with dounce for 10 strokes
#Layer the resuspended pellet on top of 10 mL 40% sucrose/HES in a 13.5 mL ultracentrifuge tube (tube = Beckman #331372)
#Top off centrifuge tube with HES until nearly full
#Spin in ultracentrifuge with rotor SW41 (saltiel login = 5238) at 39,000 rpm at 4 degrees for 1 hour
#PM should appear as band at the interface of the HES and Sucrose fractions
#Remove HES buffer above PM fraction until close to PM fraction
#Collect ~ 0.8 mL PM containing fraction
#Add 2.6 mL of HES to dilute sucrose
#Spin in ultracentrifuge with TLA 100.3 rotor at 35,000 rpm for 20 minutes at 4 degress (Tube = Beckman #349622)
#Discard supernatent
#Pellet = purified plasma membrane

3T3-L1 Plasma Membrane Isolation

2011-01-21T20:38:37Z

Buchnerd:

#Wash cells 3x in cold HES (20 mM Hepes, pH 7.4; 1 mM EDTA, 255 mM Sucrose)
#Dry cells well and add 0.6 mL HES (w/Protease Inhibitor) per 150 mm dish
#Scrape cells and homogenize with dounce homogenizer for 20 strokes
#Centrifuge @ 3,000 rpm (800g) at 4 degrees for 10 minutes
#Collect supernatant and spin @ 3,000 rpm at 4 degrees for 5 minutes
#Collect supernatant and spin in TLA 100.3 rotor at 19,000 rpm for 20 minutes at 4 degress (Tube = Beckman #349622)
#Wash pellet in 1.0 mL HES and spin in TLA 100.3 rotor at 19,000 rpm for 20 minutes at 4 degress (Tube = Beckman #349622)
#Resuspend pellet in 1.0 mL HES and homogenize with dounce for 10 strokes
#Layer the resuspended pellet on top of 10 mL 40% sucrose/HES in a 13.5 mL ultracentrifuge tube (tube = Beckman #331372)
#Top off centrifuge tube with HES until nearly full
#Spin in ultracentrifuge with rotor SW41 (saltiel login = 5238) at 39,000 rpm at 4 degrees for 1 hour
#PM should appear as band at the interface of the HES and Sucrose fractions
#Remove HES buffer above PM fraction until close to PM fraction
#Collect ~ 0.8 mL PM containing fraction
#Add 2.6 mL of HES to dilute sucrose
#Spin in ultracentrifuge with TLA 100.3 rotor at 35,000 rpm for 20 minutes at 4 degress (Tube = Beckman #349622)
#Discard supernatent
#Pellet = purified plasma membrane

3T3-L1 Plasma Membrane Isolation

2011-01-21T20:37:31Z

Buchnerd:

#Wash cells 3x in HES (20 mM Hepes, pH 7.4; 1 mM EDTA, 255 mM Sucrose)
#Dry cells well and add 0.6 mL HES (w/Protease Inhibitor) per 150 mm dish
#Scrape cells and homogenize with dounce homogenizer for 20 strokes
#Centrifuge @ 3,000 rpm (800g) at 4 degrees for 10 minutes
#Collect supernatant and spin @ 3,000 rpm at 4 degrees for 5 minutes
#Collect supernatant and spin in TLA 100.3 rotor at 19,000 rpm for 20 minutes at 4 degress (Tube = Beckman #349622)
#Wash pellet in 1.0 mL HES and spin in TLA 100.3 rotor at 19,000 rpm for 20 minutes at 4 degress (Tube = Beckman #349622)
#Resuspend pellet in 1.0 mL HES and homogenize with dounce for 10 strokes
#Layer the resuspended pellet on top of 10 mL 40% sucrose/HES in a 13.5 mL ultracentrifuge tube (tube = Beckman #331372)
#Top off centrifuge tube with HES until nearly full
#Spin in ultracentrifuge with rotor SW41 (saltiel login = 5238) at 39,000 rpm at 4 degrees for 1 hour
#PM should appear as band at the interface of the HES and Sucrose fractions
#Remove HES buffer above PM fraction until close to PM fraction
#Collect ~ 0.8 mL PM containing fraction
#Add 2.6 mL of HES to dilute sucrose
#Spin in ultracentrifuge with TLA 100.3 rotor at 35,000 rpm for 20 minutes at 4 degress (Tube = Beckman #349622)
#Discard supernatent
#Pellet = purified plasma membrane

3T3-L1 Plasma Membrane Isolation

2011-01-21T20:35:37Z

Buchnerd: Created page with '#Wash cells 3x in HES (20 mM Hepes, pH 7.4; 1 mM EDTA, 255 mM Sucrose) #Dry cells well and add 0.6 mL HES (w/Protease Inhibitor) per 150 mm dish #Scrape cells and homogenize with...'

#Wash cells 3x in HES (20 mM Hepes, pH 7.4; 1 mM EDTA, 255 mM Sucrose)
#Dry cells well and add 0.6 mL HES (w/Protease Inhibitor) per 150 mm dish
#Scrape cells and homogenize with dounce homogenizer for 20 strokes
#Centrifuge @ 3,000 rpm (800g) at 4 degrees for 10 minutes
#Collect supernatant and spin @ 3,000 rpm at 4 degrees for 5 minutes
#Collect supernatant and spin in TLA 100.3 rotor at 19,000 rpm for 20 minutes at 4 degress (Tube = Beckman #349622)
#Wash pellet in 1.0 mL HES and spin in TLA 100.3 rotor at 19,000 rpm for 20 minutes at 4 degress (Tube = Beckman #349622)
#Resuspend pellet in 1.0 mL HES
#Layer the resuspended pellet on top of 10 mL 40% sucrose/HES in a 13.5 mL ultracentrifuge tube (tube = Beckman #331372)
#Top off centrifuge tube with HES until nearly full
#Spin in ultracentrifuge with rotor SW41 (saltiel login = 5238) at 39,000 rpm at 4 degrees for 1 hour
#PM should appear as band at the interface of the HES and Sucrose fractions
#Remove HES buffer above PM fraction until close to PM fraction
#Collect ~ 0.8 mL PM containing fraction
#Add 2.6 mL of HES to dilute sucrose
#Spin in ultracentrifuge with TLA 100.3 rotor at 35,000 rpm for 20 minutes at 4 degress (Tube = Beckman #349622)
#Discard supernatent
#Pellet = purified plasma membrane

3T3-L1 Adipocyte Fractionation

2011-01-18T15:52:29Z

Buchnerd: /* Fractionation */

__NOTOC__
==Materials==
*PBS
*Lysis Buffer (25 mM HEPES, 250 mM Sucrose, PI tablet)
*Optiprep
*Cool centrifuges and rotors for JA17/JA25.5, TLA100.3 and NVT90 to 4C

==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 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 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 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.
#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==
#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 '''do not boil if probing for GLUT4'''

==References==
PMID 17765682