Hi all, I am validating a polyclonal antibody raised against human ANT4, through WB and ICC. In most of my controls, the antibody shows very little off-target binding, but when I pre-incubate the antibody with the immunogenic peptide which was used the raise the antibody, then perform ICC in HEK293 cells, I see strong, blobby nuclear staining, except in cells undergoing mitosis, where the staining diffuses throughout the cytoplasm. HEK293 cells don't express my protein of interest, and without the blocking peptide show only moderate levels of random punctate cell-surface staining. Can anyone explain the molecular mechanism or significance of what might be going on? Thanks! I can upload photos if that is helpful.
So, if I have understood correctly, your antibody does not react much with the HEK293 cells, and is not expected to. But when you add the peptide the antibody was raised against you do get staining.
The step of adding the blocking peptide is usually used in order to see whether staining that you DO get with just the antibody is a specific reaction. What you expect to see is that specific staining is eliminated ( or at least reduced) by competition with free peptide.
In this case it looks likely that adding the peptide causes a conformational change in the antibody ( or possibly in some protein in the cell, but most likely - the antibody) and that conformation change just happens to cause the antibody to bind to something in the cell.
If there is no specific staining with the antibody by itself you should not expect a reduction. That you get an increase may just be bad luck because of some random protein that is in HEP293 cells . Or is it possibly because you increased the amount of antibody you used - expecting to need a lot because there was no intrinsic protein for it to bind to and wanting to show up even minor amounts if they were there - so that what you are getting is more about aggregation/precipitation of the antibody once the peptide is there - a non-specific effect- maybe the precipitate likes to stick to DNA/histone complexes or similar. Just a thought. If so there could be something somewhere in the literature about this being quite common if you use high concentrations of antibody.
Thanks for the thoughtful answer! You've understood correctly. I suppose there was no real conceptual reason to do a blocking condition on my negative control cells, but I did it out of habit and it gave an unexpectedly interesting result. I've never heard of an antigenic epitope changing the conformation of an antibody before, but I suppose it's possible! In the target cells (human sperm) the blocking does in fact decrease on-target fluorescence, while also strangely increasing signal outside of the cells, perhaps targeting some residual protein on the cover slip or something in the mounting media.
In the HEK cells, the blocked binding seems to be some kind of nucleoprotein, because it doesn't colocalize with dapi in cells in interphase or in mitosis. I didn't increase the antibody concentration to compensate for the blocking, which made it seem extra strange!
Are your imaging conditions equal for the positive cells and the negative controls? I ask because it could be that you are observing the background (unspecific) fluorescence in your blocked antibody. If you increase the laser intensity/exposure in your positive ICC, can you see that nuclear staining? (Could be a lot more dim because your specific staining exhaust the ab)
Finally, I have observed that some ab induce diffuse unspecific staining in tissue sections when there isn't epitopes for the ab in the sample, could that be related? 🤷
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