If the cells are neither fixed nor permeabilized, can a protein specific antibody bind and can an Alexa based fluorophore recognize it? Can live cell imaging work without a GFP-fusion protein or any tagged protein for that matter?
Hi there,
i think that either the epitope has to be on the cell surface in this case that can be a way to detect it. But if the protein is not expressed high enough, the siganl might be pretty small. i usually fix cells directly after calcium imaging. (~ 0.5-2 min) thereby degradation effects are not to harmful..... please contact me if want more details
Hi,
i agree with Rene, in using antibody to detect surface epitopes (people use it in FACS as well). to detect cytosolic proteins, it will be a hurdle to get the antibody in without permeabilizing the cell. i would suggest liposomes carrying antibody, but its of low efficiency and you need a lot for limiting amounts inside the cell.
I agree that if your protein is cytoplasmic, immunoprobing without fixation to stabilize and without extraction to permeabilze should be rather impossible. Depending on the protein though you may find specific fluorescently tagged molecules that are both cell permeable and adequate to label in living cells. Such options though are very limited. If you wish to avoid GFP-tags due to their size you can clone your protein with a small tag like a tetracysteine motif which can then be tracked with biarsenical fluorescein or rhodamine analogues, such as FlASH and ReASH. For any kind of solution you are looking for, it is best to start at the Invitrogen homepage (www.invitrogen.com) whereby you will find numerous fluorescent solutions to your problems.
Let me clarify that my protein is both cytoplasmic as well as membrane bound. It reacts to ligand stimulus and expression increases on membrane post ligand binding. Its a post synaptic protein.
Right now I am doing imaging to capture membrane part but nit live, 2% PFA fixation and non-permeabilizing conditions. I want to do a quick check for live cell. So was curious whether antibodies be used or surely an FP protein is needed like GFP or CFP-protein.
Be also careful that adding a bulky tag ad an antibody to a protein of some tens of kDa might alter its function and or localization in live cells...apart from that, if the protein exposes the epitope to which your ab binds in the extracellular space, the labellinng might work.