Chromatin immunoprecipitation (ChIP): Revisiting the efficacy of sample preparation and sonication, and of sheared DNA extraction, quantification, and analysis via polymerase chain reaction (PCR)

Molecular biologists routinely use ChIP as a tool to study protein‐DNA interactions in healthy and diseased systems. However, the assay can be cumbersome and fraught with technical error. The objective of this work was to develop an easier ChIP protocol and an accurate method of quantifying the final ChIP product. We demonstrate that harvesting with a detergent‐based buffer improves the rate of fixed‐cell recovery; that ChIP lysis buffers do not lyse fixed cells; and that sonication intensity affects fixed‐cell DNA recovery. We confirm that DNA can be extracted from antibody‐protein‐DNA complexes rapidly and easily using chelex‐100 but we find that the final samples are too dilute for evaluation of DNA shearing or for DNA quantification via nanospectro‐photometry. However, DNA extracted from the Mock‐ChIP supernatant via the phenol‐chloroform‐isoamyl alcohol method can be used to evaluate DNA shearing efficiency by gel electrophoresis and used as the standard in a PicoGreen assay. This enables accurate quantification of DNA in chelex‐extracted ChIP samples. Thus, ChIP samples can be normalized to DNA concentration prior to performing PCR. These data led to the development of a QUICK ChIP protocol that can be completed in nine bench hours over two days; a rapid, accurate and repeatable way to quantify ChIP DNA; and PCR data that more accurately depicts treatment effects on the protein‐DNA interactions of interest.