Review this powerful technique, get advice on selecting antibodies, and learn troubleshooting techniques with our Epigenetic specialist
ChIP is a very powerful technique that allows for the analysis of protein localization on DNA regions in the genome. Selective enrichment of a chromatin fraction using antibodies allows for the isolation of DNA regions and interacting proteins.
ChIP protocol: chromatin preparation, sonication, immunoprecipitation, cross-linking reversion and DNA purification, DNA analysis, needed controls
About the Presenter:
David Grotsky received his PhD in Molecular Genetics and Genomics
from Washington University in St. Louis. His PhD thesis focused on
studying the regulation of DNA repair factors and how they impact
genomic stability and proliferation in tumor cells.
David is currently the Epigenetics Specialist on Abcam’s Scientific Support Team.
Now, I will discuss the protocol in more detail, indicating which stages are crucial and where special attention and care has to be taken when performing the chromatin immunoprecipitation.
When using X-ChIP we start with cross-linking of the antigen of interest to its chromatin-binding site. Histones themselves generally do not need to be cross-linked, as they are already tightly associated with the DNA. Other DNA binding proteins that have a weaker affinity for DNA may need to be cross-linked. This holds them in place and avoids protein dissociating from the chromatin-binding site. The further away from the DNA your interaction of interest lies then the less effective ChIP will be without cross-linking.