Antibodies may have different characteristics in regards to affinity, avidity, HIER needed or not, useable on fixed tissue or not. And maybe an overall quality, when the product itself is not really good.

Are your two antibodies comparable in all aspects? Do both work on positive controls in expected manner? The bound epitopes may be differently sensitive to fixation....

Antibodies may have different characteristics in regards to affinity, avidity, HIER needed or not, useable on fixed tissue or not. And maybe an overall quality, when the product itself is not really good.

Are your two antibodies comparable in all aspects? Do both work on positive controls in expected manner? The bound epitopes may be differently sensitive to fixation....

I agree with Gudrun Lang. We used two polyclonal antibodies and got totally different results. In our case, both should have shown cytoplasmic staining, but we ended up with one nuclear, even on controls and repeated staining.

A lot depends on the peptide against which the antibody is raised.

Obviously. I think that the described result is also the proff that both te antibodies are working well

Greetings

Having proper positive and negative controls that react as required are the best way to control the quality of your experiment. So standardize the conditions for both, the sample as well as the methodology, and it should resolve your problem.

The response to your original question therefore is that though theoretically they should be similar but depending which regions of the GPCR the antibody is raised to and if that region is available for binding with antibodies they may not.

Pasha Grachev does have a very good point about internalization of the protein.

Good luck. God bless.

I agree, all staining must be performed on as identical sections as possible. With serial sectioning of the same animal this should not be a problem. Theoretically, yes, it shouldn't matter what antibodies you use, the staining pattern should be identical. The distance between epitopes is considerably smaller than the wavelength of light so staining should be identical. In practice however, things are not as straightforward. You need to know where the epitopes are in the protein. It may be that one antibody recognizes an epitope that is hidden at the plasma membrane for example (if the protein binds an accessory protein at the surface), and so will not stain the cell membrane, but may stain protein in the biosynthetic pathway, when the epitope is exposed. Have you tried expressing the protein in a cell that normally doesn't express the protein?

If your antibodies are binding the same protein, irrespective of which epitope they recognize, they ought to colocalize when you do IF. If you are not seeing this then it can only mean that your a.b. is not specific enough to its supposed epitope, i.e., you have a crappy a.b., or it could also be that one a.b. binds to an epitope that is part of a conserved domain found in other proteins.

I suggest to verify the co-localization by using confocal microscopy. This method may accurately confirm your finding.

Results may be same or may be different, and depend on the epitope on GPCR against which the antibodies are raised. Generally antibodies raised against C-terminal epitope/domain give cytosolic, and the antibodies raised against N-terminal epitope/domain give nuclear or membranous staining or localization. This is because N-terminal region signals proteins to their target (membrane or nucleus) from cytosol, the site of their synthesis. As suggested by Dr. Ierardi Enzo, confocal microscopy will clearly tell about the location and its functional significance.