Imaging of GFP - protein

[breathoffire]

I want to know what is the best way to go about imaging the migration of a large transmembrane protein from its membrane bound form to it's intercellular form in the nucleus? This protien is huge (~300kDa) at full legnth. It does go through post-transcriptional modification, which is what allows it to move from the membrane to the nucleus. I am trying to prove that this protien has some regulatory function in addition to being a membrane bound protien. Any suggestions would be great.

Right now I am microinjecting the cells with a GFP construct and imaging them with a fluorecence scope.

[Warthaug]

I believe I can lend you some help here, but I need some details first:

1) Is this a transmembrane protein, or a soluble protein?
2) If transmembrane, is part of it ever exposed to the outside of the cell.
3) Do you have access to an antibody that recognizes your protein in the unmodified and post-TL modified state?
4) Do you have access to a scope with photoactivation capabilities?
5) Is this protein expressed naitivly in your cell type?

Bryan

[breathoffire]

Bryan[/QUOTE]

Sorry this took me so long to get back to. I had another project that this kind of took the back burner to.

1) Is this a transmembrane protein, or a soluble protein?

It is actually both - when it is in it's full legnth form it contains a transmembrane domain, but then it goes through post translational modification where it is cut by an enzyme controlled by hormones. It is then thought to move into the nucleus where it has effects on transcriptional processes.

2) If transmembrane, is part of it ever exposed to the outside of the cell.

Yes, there is a small portion outside of the cell, but this part is not attached to the protien protion that moves through the intracellular matrix to the nucleus.

3) Do you have access to an antibody that recognizes your protein in the unmodified and post-TL modified state?

Currently, we have the monoclonal antibody that recognizes only the full legnth form. We do have the polyclonal antibody that will recoginze all three forms of the protien (full legnth, transmembrane, and intracellular).

4) Do you have access to a scope with photoactivation capabilities?

Yes, I am sure that we do even though I have no idea what this is

5) Is this protein expressed naitivly in your cell type?

Yes, this is a ubiquidously and highly conserved protien.

Thank you for your assistance.

Bryan[/QUOTE]