December 1, 2020, 10 – 11 am, Zoom Webinar, GRCF Webinar Series featuring Cell Signaling Technology, ‘ Analysis of Epigenetic Marks and Mechanisms in Disease: The Importance of Highly Validated Antibodies & the CUT&RUN Assay’

Abstract:

Research in the field of epigenetics has grown at a rapid pace since the discovery of the first histone acetyltransferase enzymes over 20 years ago. Since then, significant advances have been made in our understanding of the basic epigenetic mechanisms regulating gene expression and genomic stability, and the impact of epigenetic deregulation on cancer and other diseases. Much of our knowledge of these mechanisms comes from the utilization of antibodies to probe the protein levels and localization of transcription factors, chromatin regulators and histone modifications in different cell types and tissues. While antibodies have been a key reagent driving advancements in epigenetic research, there are increasing numbers of publications raising concerns about the quality of the antibodies being used. Recent advancements in antibody technologies, specifically the development of rabbit monoclonal antibodids presents solutions to many of these problems. I will demonstrate how the utilization of rabbit monoclonal technology combined with thorough antibody validation can lead to generation of high quality rabbit monoclonal antibodies that show exquisite specificity, sensitivity, and reproducibility across multiple applications.

     Over the years the chromatin immunoprecipitation (ChIP) assay has contributed greatly to our understanding of epigenetics, providing us with an abundance of data regarding the localization of histone modifications and protein binding across the genomes of a multitude of organisms. However, this assay has a number of limitations that have restricted the types of cells and tissues that can be analyzed. In addition, not all transcription factors and cofactors have been amenable to study in the ChIP assay. Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is a new technique developed to overcome many of these limitations. I will introduce the CUT&RUN assay, discuss key differences between ChIP and CUT&RUN, and demonstrate how CUT&RUN paired with highly validated antibodies can be used to map histone modifications and protein binding in less time, using fewer cells, and with less background than the ChIP assay.