Organelle separation - buffer, homogenization, centrifugation force? - (Oct/23/2007 )

Hi all good people,
I would have questions (again) regarding cell organelle separation.
My procedure of homogenization and separation:
I use phosphate buffer saline (PBS) (pH=7.2) or sometimes 20mM Tris/HCl + PBS (1:1, pH=7.5) with 0.25M sucrose + protease inhibitors. I homogenize (gently) cells in this buffer with Potter-Elvehjem homogenizator (glass cylinder with tefflon pestle – something like Dounce homogenizator) with 20 strokes - in microscope I can see many non-disrupted (probably) cell nuclei after the homogenization. Then, I centrifuge at 1020xg,15min then 10800xg, 15min then 20200xg, 15min then 110 000xg, 60min.

I want to have as highest as possible amount of plasma membrane and endoplasmic reticulum in pellet of 110 000xg.
The questions:
Is/are "all" plasma membrane and endoplasmic reticulum pelleted at 110 000xg or will be any substantial (big) part of these organelles in pellet of 20 200xg?
Should I use more aggressive homogenization to disrupt membrane in smaller peaces to pellet it at 110 000xg better?
If I would suppose that nuclei are in 1020xg pellet, mitochondria in 10800xg, what is in 20 200xg (cytoskeleton, Golgi vesicles or. . .)?

Thanks for your advices!
Vic

Have you checked your fractions? Are you sure that you will get the PM and ER at the last spin???? I would suggest double check, looking for 'subcellular fractionation', PubMed will have lots of good papers for this. I recommend David James' group's works, but there are undoubtedly more. Briefly, the PM should come down at 13-17Kg. ER and small fraction of PM is considered high-density membrane, will come down at about 30-38Kg. The vesicles are low density membrane and come down at the highest spin, about 100Kg. There could be contamination between the fractions, which you will have to work out the best procedure to minimize, but the main fractions are roughly like that.