3T3-L1 Adipocyte Fractionation: Difference between revisions
modified volumes to reflect whether using optiprep or not. |
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#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== | ||