I want to visualize a protein which will reach the plasma membrane and localize on the extracellular space while performing live cell imaging. Is there a way to have a tag, which will only become fluorescent when it reaches the extracellular space?
Hi,
Are you recombinantly expressing the protein inside the cell?
Dirk
Yes. I could potentially have a tag which would become fluorescent only in the extracellular space.
Ok, there are some GFP protein tags that changes their fluorescent intensity depending on the environment they're in. Or you could permeabilize the cells and use antibodies (with fluorescent tag) to see where you're proteins is inside the cell? What type of microscope are you using?
You can use a pHulorin as a tag. Is a pH-sensitive GFP; its fluorescence is very low at acidic pH (5.5, into a lysosome or a synaptic vesicle) but recover its fluorescence at normal pH (7.4, the extracellular space)
Thank you for the help. The problem is that this protein is not secreted into vesicles but it is just translocating across the plasma membrane. So the ecliptic pHluorin could not be used in this case. Is there any relevant GFP variant which would change its fluorescent properties extracellularly? I can already detect the protein with antibody staining but unfortunately this can not be used for live cell imaging.
Hi Eleni, although I have never used them myself and cannot tell you how good they work, the SNAP/CLIP tags from NEB might be usefull in your case. After addition of a non-cell permeable substrate (see SNAP Surface) one should be able to do live cell imaging and visualize only the translocated protein (assuming that the tag is also presented to the outside). I would be happy to learn what did the job for you in the end.
Thanks Anja! This is an interesting idea indeed! I will let you know what will work in the end.Thank you for the help!
Hi Eleni,
Another alternative, really easy is to label your plasma membrane and see where your protein (GFP-tagged for example) is seen in comparison with your labelled plasma membrane.
You can label the plasma membrane (and all membranes) with FM dyes : there is several of them and have different properties that could fit with your problematic.
Hope it will help you.
@ Lucille, in general the idea to stain the PM with FM is a very good one. But you should keep in mind that you need a microscope with a best spatial resolution possible (superresolution?) in order to distinguish between membrane staining and a membrane spanning protein-XFP fusion. In addition, and this is very important, the objective needs to be corrected for chromatic abberation of GFP and RFP.
I would suggest, since it was not mentioned yet, to use redox-senstive GFP (roGFP). To be honest, I don't know how the redox-situation in animal cells and their extracellular space is. But maybe you have a look at the publication in NatMeth. In plants it was also used to distinguish lumenal or cytosolic orientation of membrane proteins.
http://www.nature.com/nmeth/journal/v5/n6/abs/nmeth.1212.html
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