membrane isolation protocol - (Apr/27/2007 )

Hi!

Could someone show me a membrane isolation protocol?? I need to test if PI3K translocate to plasmatic membrane within my treatments and I have thought to see it using confocal microscopy and isolating membrane...
Thank you for your help!!!

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Confocal micros. an isolating membrane? You meant 2 approaches, right? As in checking by confocal (IF or living cells) and checking by isolating membrane (IP, WB...)

For isolating PM, check out David James GLUT4 papers, they did that alot, using differential centrifuge. Briefly:
- Rinse and then homogenize cells in ice-cold HES (20mM Hepes pH7.4, 1mM EDTA, 250mM sucrose) (use homogenizer)
- Spin at 17.3Kg, 20min, 4oC
- Collect the pellet, wash and resuspend in 1ml HES
+ layer onto a 1.12M sucrose cushion (prepare in 20mM hepes pH 7.4, 1mM EDTA)
+ spin 100Kg, 1h, 4oC
+ look at the interface between the two phases, PM will form a layer above the cushion. Get it out, normally by syringe
- The supernatant of the first spin:
+ spin 38.7Kg, 20min -> pellet: ER and small pieces of PM

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