ChIP Assay Using Chromatin Isolated From IMR90 Cells. The PureEpi Chromatin Preparation and Optimization Kit is designed to generate chromatin from both cell lines and primary tissues. To demonstrate how this can be done with minimal protocol changes, IMR90 cells were prepared and analyzed by ChIP. ChIP experiments were performed using ChIPAb+ Phospho-CREB (Ser 133) (cat # 17-10131) and the Magna ChIP™ HT96 Chromatin Immunoprecipitation Kit (cat # 17-10077). ChIP results were analyzed using qPCR and the ΔΔ Ct method to calculate fold enrichment versus an IgG control at the positive locus. The positive/negative calculation was figured using the CREB signal at the positive locus and negative locus in relation to the input. The results of the five minute sonication, which gave the best performance, are presented here. The mouse primers used were positive locus mouse GAPDH promoter, negative locus mouse Myo D promoter.

Variation in ChIP results using chromatin fragmented under different sonication settings including Time ON/OFF, Power, and Cycle Number. A ChIP experiment using ChIPAb+ Phospho-CREB (Ser133) (cat # 17-10131) was used to analyze the chromatin, using the Magna ChIP™ HT96 Chromatin Immunoprecipitation Kit (cat # 17-10077). ChIP results were analyzed using qPCR and the ΔΔ Ct method to calculate fold enrichment versus an IgG control at the positive locus.

ChIP Assay Using Chromatin Isolated From Mouse Liver Tissue. The PureEpi Chromatin Preparation and Optimization Kit is designed to generate chromatin from both cell lines and primary tissues. To demonstrate how this can be done with minimal protocol changes, mouse liver tissue was prepared and analyzed by ChIP. ChIP experiments were performed using ChIPAb+ Phospho-CREB (Ser 133) (cat # 17-10131) and the Magna ChIP™ HT96 Chromatin Immunoprecipitation Kit (cat # 17-10077). ChIP results were analyzed using qPCR and the ΔΔ Ct method to calculate fold enrichment versus an IgG control at the positive locus. The positive/negative calculation was figured using the CREB signal at the positive locus and negative locus in relation to the input. The results of the five minute sonication, which gave the best performance, are presented here. The mouse primers used were positive locus mouse GAPDH promoter, negative locus mouse Myo D promoter.

Typical Chromatin Preparation Workflow. Starting with a cell or tissue sample, proteins and DNA are cross linked together using formaldehyde. Cells in the sample are then lysed to isolate the nuclei. Once the nuclei have been lysed, the extracted chromatin is sonicated into fragments that are suitable for ChIP analysis.