plasma membrane-selective permeabilization?
Ian A. York
iayork at panix.com
Tue Nov 26 11:07:42 EST 1996
In article <57ec7d$jqt at netty.york.ac.uk>, <gst3 at york.ac.uk> wrote:
>I'd be very grateful if anybody could point me in the direction of techniques to
>specifically permeabilize plasma membranes in cultured mammalian cells whilst
>leaving as much else (mito, lysosomes etc) unaffected.
I would also be interested in knowing other people's experiences.
I've been trying to do this for some time now. We started out with
streptolysin O, based on some published articles which seemed to show that
it was pretty specific for the plasma membrane (the approach was to allow
the SLO to bind at <4 oC, wash away any non-bound SLO with cold buffer,
then briefly shift to 37 oC to allow the bound stuff to form pores).
Unfortunately, after we'd been working with it for a while, we realized
that the permeablization was *not* specific to the plasma membrane. I've
since spoken to someone else who has seen the same thing and demonatrated
it (unpublished) in a more rigorous and quantitative way than our approach
(which basically involved saying, "Hey! That looks bad!).
On this guy's recommendation, we then turned to using saponin at low
concentrations: 0.0075% for one of the cell lines we're looking at,
0.0125% for the other. This does seem to be specific for the plasma
membrane as far as we can tell (and again it's been looked at more
carefully by the other group). However, the phenomenon we're looking at
doesn't seem to be happening with the saponin, which may mean that it's an
artifact of the permeablization (though actually it's hard to think of a
way that permeablization of internal membranes could lead to this
particular artifact) or which may mean that the pores the saponin
generates are too small for our purposes. Saponin is supposed to make
pores lage enough for antibody (~150 kDa) and we have indirect evidence
that the pores may be considerably larger, but nevertheless we're not
entirely happy with it.
We haven't yet tried digitonin, but we might still try it. I don't
believe that digitonin is all that specific for the plasma membrane - eg
you can use digitonin to permeablize cells and get out ER proteins - so
it's a matter of titrating concentration util you find the right one, as
we did for the saponin.
I'd appreciate knowing about other approaches, and any tips or tricks that
have worked for you.
Ian York (iayork at panix.com) <http://www.panix.com/~iayork/>
"-but as he was a York, I am rather inclined to suppose him a
very respectable Man." -Jane Austen, The History of England
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