Antibody-trapping presents a widespread pitfall for microscopy and genomics in the nucleus

Many microscopy and genomics technologies that map chromatin proteins and modifications rely on the diffusion of antibodies (Abs) to target epitopes within whole nuclei. Here, we reveal a critical flaw in such methods that arises when Abs become trapped at the edge of nuclear structures and fail to reach internally-positioned epitopes.

This “Ab-trapping” results in artifactual peripheral signal that fundamentally distorts the apparent positions of chromatin features across the genome and nucleus. Using computational modelling and experimental validation, we demonstrate that Ab-trapping is caused by a combination of three compounding factors - high epitope abundance, high Ab affinity, and low Ab diffusion rates. Ab-trapping can thus systematically misrepresent the localization of many prevalent chromatin features like histone modifications, transcription factors, nucleolar proteins, and protein tags.