Hi!
I need to prove that my protein is localized on the cell-surface but not inside the cell in soluble form.
How can I make it?
There is an opinion to wash cells, to label all cell-surface proteins with NHS-biotin, and to collect labelled fraction of cell-surface proteins with streptavidin magnetic beads. After that I can make western blot with specific Ab.
Maybe there is another way?
Protein and cell lines are human.
Hi there,
The purpose is to show that your protein of interest is accessible without lysing the cells, right? As you have an antibody in hands, I would suggest immunofluorescence on intact cells. An other approach would be to submit cells to mild pronase digest (degrading only exposed parts of proteins) and see what happens to your protein by WB.
Actually, there is no big need to keep cells intact. I need only to prove that my protein of interest is localized on the cell-surface. The protein has two forms – it could be expressed on the cell-surface or in soluble form inside the cell. Usually both forms are present.
You could perform immunohistochemical analysis on cells that are fixed and permeabilized and compare it to another well of cells that are fixed but not permeabilized. I did something similar in my 2005 and 2007 papers if you want to find a specific protocol.
Hi again,
If the cell surface expressed form is associated tightly enough with membrane then cell fractionation should be enough to identify the two populations.
Hi Victor,
just one remark, if you decide to use biotinylation (what in my experience is the easiest way by far). Do not use NHS-biotin, it is membrane permeable. Use Sulfo-NHS-Biotin, which is not membrane permable, instead (and preferably do the experiment at 4°C to limit endocytosis; if you want a protocol, contact me).
All other methods (at least the ones I know a relative cumbersome). I have read about one method using Trypan blue to quench extracellularly flourescence. I you have an antibody which recognize your protein in vivo, you could use this technique. If you use the search term "trypan blue quenching protocol" in the web you should find something (but I think also this method is quite difficult).
Good luck,
Carsten
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