vacoular or plasma membrane protein?

Irit Rappley via methods%40net.bio.net (by irappley from scripps.edu)
Tue Mar 22 17:13:00 EST 2011


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
>
>
>
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