I can add a few details to DK's general protocol:
1. Expression in E. coli, Sf9, or mammalian cells -- whichever works
best for your protein. If your protein is glycosylated or undergoes
other post-translational modifications, you will probably need insect
or mammalian cells.
2. Detergents can be the hardest part of the assay to troubleshoot.
Try to find the mildest detergent that works for your application.
CHAPS/CHAPSO is commonly used for this type of application, but there
are many other options.
3. Having lysed your cells with the optimized detergent protocol, IP
your protein or protein complex. Again, lots of troubleshooting will
be required before this works well.
4. Prepare a film from the lipids you want to use (see http://
www.avantilipids.com/index.php?
option=com_content&view=article&id=1384&Itemid=372 for some basic
information). Add your IP'ed proteins to the hydration solution, then
sonicate or extrude to form proteoliposomes. From my experience,
detergents cannot be dialyzed out of such a system because they
"stick" to the lipids that you want to keep. A product called "bio
beads" can be used to remove the detergent if necessary. For some
applications, a small amount of detergent is acceptable and might not
interfere with the function of your protein -- or more precisely,
interference from a small amount of detergent might still leave
enough protein activity for your application.
Hope this helps.
Irit
On Jan 19, 2011, at 6:18 PM, DK wrote:
In article <[email protected]>,
Fulvio Celsi <[email protected]> wrote:
Hello to all
a question for you...so, my PI asked if it is possible and how
much is
difficult to synthetize and produce a transmembrane protein and to
insert it into a lypid bilayer. The biochemist inside me started to
scream..but my background as biochemist is quite old. So..any
suggestion on how it can be done? I saw I kit from Invitrogen
(membraneMax) that state that is quite easy (as every kit in the
world). Anyone has used it??
thanks a lot in advance! :)
Yes it is possible and it has been done many times.
1. Expression as usual (transmembrane proteins are challenging and
yields are almost always low).
2. Find high CMC detergent that preserves protein activity (may take
a lot of screening).
3. Purify (can be difficult but getting much easier with tags/
antibodies)
4. Mix with lipids and slowly dialyze out (or remove by some other
means) the detergent. Depending on conditions/lipid mixture, liposomes
of various sizes can form. Giant liposomes can be used in patch clamp
directly, others can be fused with planar bilayers or fused
together to
form larger liposomes.
No kit will ever work for all proteins. I doubt any kit can even
work for
most proteins.
DK
Fulvio Celsi
BsC,PhD
Sector of Neurobiology,
International School for Advanced Studies,
Scuola Internazionale di Studi Superiori Avanzati (SISSA),
Via Bonomea 265 34136
Trieste
Italy
Office: +39 0403787 771
Mobile: +393286489131
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