Hello, I was wondering if anyone has a good protocol for inner and outer membrane isolation. I need to detect a Histaged protein on the inner membrane. I have an outer membrane protein preparation protocol, but not an inner membrane isolation protocol.
I know also that it involves French press and the alternative could be Sonication. I don't have a French press available at the lab, but there is a sonicator, however I don't have a protocol for it. Does any one have of or know where to get one?
At the same time, does anyone have other suggestion?
Thank you ver much
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I don't have a protocol for you but 3 suggestions:
a) if you can prep away the outer membrane and your protein is still
there, then this will be a hint (but no proof as there is lots of
space inside a cell) for a location at the inner membrane. you should
test for control proteins with known location, however. Maybe you can
apply a membrane preparation (preparative ultracentrifugation with
density gradient probably) protocol after you have removed the outer
membrane to recover the inner membrane.
b) if you have an antibody which detects your protein in native state,
you could demonstrate the location of your protein by labeling intact
and permeabilized / fixed cells by immunocytochemistry or FACS. I
think, for His-tags, suitable antibodies should be available from many
c) likewise, if your protein is outside, you might digest it away with
eg trypsin and demonstrate that it is gone (and maybe that it returns
after some time by biosynthesis).
d) very hypothetical: If you can get your protein into erythrocytes,
there are protocols for "inverting" their membrane. If I remember
correctly, this is sort of hypotone lysis. Maybe this can be done even
forth and back, so you could check if your protein binds to inverted
erythrocytes, but not to intact erys.
On Sep 26, 9:54 pm, Natalia Oliveira <[Only registered users see links. ]> wrote:
Hello, I was wondering if anyone has a good protocol for inner and
outer membrane isolation. I need to detect a Histaged protein on the
inner membrane. I have an outer membrane protein preparation
protocol, but not an inner membrane isolation protocol.