Hi Emad,
Overexpression of any protein can often lead to non-natural
localization. It's possible that the cells are just expression so much
of your protein that it has nowhere to go except vacuoles. If you want
to know where your protein naturally exists, then you need to work with
endogenous protein.
An easy way to know whether your protein is on the plasma membrane is to
do a bioinylation assay. Basically, you add soluble
(membrane-impermeable) biotin to your cells so that it only labels
proteins on the outside of the membrane. Then you lyse your cells and
detect with avidin. Only extracellular proteins will be detected, and
you can see whether yours is one of them. Pierce sells lots of
biotinylation kits for just this sort of assay.
You could also try biochemical fractionation. Here, you would homogenate
your cells in detergent-free buffer and then use different
centrifugation speeds to separate various subcellular compartments. This
is never 100% clean, but you should be able to separate large plasma
membrane sheets from vacuoles and see where your protein is enriched.
Again, though, any work you do should be using endogenous protein only.
Save the labeled and overexpressed protein for later assays, when
localization is not relevant.
Hope this helps,
Irit
On 3/22/2011 4:54 AM, Emad ALBAROUKI wrote:
Hello everybody,
I have a question concerning a plasma membrane protein, which have been
expected using different proteomic software to be localized on the plasma
membrane.
After tagging with gfp it looks to be localized in more than 80% on vesicle
structures which we assumed to be vacuoles.
Is it possible to be a bifunctional protein such as transporter on plasma
membrane and vacuole? or that could be an artifact of gfp fusion process?
Is there any programs or software can discriminate between both
compartment's proteins?
Thank you for your help in advance
Emad
_______________________________________________
Methods mailing list
[email protected]
http://www.bio.net/biomail/listinfo/methods
_______________________________________________
Methods mailing list
[email protected]
http://www.bio.net/biomail/listinfo/methods
