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I have another question. Is this antibody more sensitive to the antigen in the nucleus? In my IHC staining, I found that the signal was stronger in the nucleus than the cytoplasm within both of my target tissue and its surround tissues. The staining signals in cytoplasm look like the background. Do you have any suggestion about that?
Asked on Jun 08 2013
We do not have any data suggesting that the antibody has higher affinity for nuclear PSMA3 than for cytoplasmic PSMA3. The differences in staining intensity may be due to unequal distribution of the protein (more PSMA3 in the nucleus than in the cytoplasm), or the epitope that the antibody recognizes on PSMA3 may be obscured in the nucleus, depending on what it is complexed with, compared to what it is complexed with in the cytoplasm.
A western blot of denatured subcellular fractions might help clarify which is the case. The antibody is raised against a peptide sequence which, after denaturation of the samples, will be available for the antibody to bind to, without interference from proteosome complexes that are unique to the nucleus or cytoplasm.
On the other hand, the antibody may simply be cross-reacting with something other than PSMA3. It is, however, expected to be present in both the nucleus and the cytoplasm., and what apppears to be cytoplasmic background may in fact be PSMA3.
Answered on Jun 08 2013