6
edits
Changes
→Fractionation
__NOTOC__
==Materials==
*PBS
#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==
#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 boilif probing for GLUT4'''
==References==