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.

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