3T3-L1 Adipocyte Fractionation: Difference between revisions

Line 11:

#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 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.

#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==

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#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'''

#Store samples at -80. Make up SDS samples and '''do not boil if probing for GLUT4'''

==References==

PMID 17765682

@@ Line 11: / Line 11: @@
 #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 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.
-#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==
@@ Line 22: / Line 22: @@
 #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'''
+#Store samples at -80.  Make up SDS samples and '''do not boil if probing for GLUT4'''
 ==References==
 PMID 17765682