Subcellular Quantitation of ADP-Ribosylation by High-Content Microscopy

ADP-ribosylation is a posttranslational modification with many functions ranging from the DNA damage response to transcriptional regulation. While nuclear ADP-ribosylation has been extensively studied in the context of genotoxic stress mediated by PARP1, signaling by other members of the family and in other cellular compartments is still not as well understood. In recent years, however, progress has been made with the development of new tools for detection of ADP-ribosylation by immunofluorescence, which allows for a spatial differentiation of signal intensity for different cellular compartments. Here, we present our method for the detection and quantification of compartment-specific ADP-ribosylation by immunofluorescence and show why the engineered macrodomain eAf5121 might be the best tool to date.