separation of cardiac endosomes from plasma membrane

Will Fuller will.fuller at kcl.ac.uk
Mon Jun 7 15:50:47 EST 1999


Hi, I wonder if you can help, as I'm at my wits end.

I'm trying to separate membrane fractions obtained from whole rat
hearts, specifically the plasma membrane (sarcolemmal) and endosomal
fractions. Although the literature suggests this should be possible by
density through a sucrose gradient (at least it is so in membranes from
other tissues), I have had no success in identifying membrane fractions
from gradients in which my plasma membrane marker (sodium potassium
ATPase) or endosomal marker (rab4) proteins are differentially enriched.
They always appear together in membrane fractions from gradients. I have
tried gradients ranging from 27% to 70% sucrose with just about
everything in between.

I can be sure that the homogenisation procedure I've adopted is pulling
all the sodium pump and rab4 off the cytoskeleton and whatever is left
behind in a low speed pellet (5000g), into a high speed pellet (240000g)
which is total cardiac membranes. Nor do I have any success if I try
separating membrane fractions by spinning at different speeds
(everything between 5000g and 240000g and even a bit faster) and looking
at the different fractions then. I am really at a loss and don't see
much in the literature to help. My specific questions are:

1.	Is there a better endosomal marker than rab4 (I've tried rab5 but
can't see it on Westerns from the heart)?
2.	Is there a better sarcolemmal marker than the sodium pump?
3.	Is it just that these membrane fractions cannot be distinguished
either by density or sedimentation co-efficients?
4.	I homogenise for 1 minute. This is necessary to dissociate all
membranes from the cytokeleton, but could it be damaging my membranes
such that any differences between them are blurred? If I homogenise for
less time the amount of membrane protein I obtain is reduced.
5.	Any references you can point me to or experiences you can share
either with cardiac or other muscle (or indeed with any tissue) would be
greatly appreciated.

Please mail me direct. Thanks in advance for your help


Will



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