Benefits of Enzyme-based Chromatin Digestion

New SimpleChIP^® Enzymatic Chromatin IP Kits from Cell Signaling Technology (CST) are co-developed by CST and NEB scientists and therefore contain the highest quality research reagents. These kits are available with either Protein G agarose or Protein G magnetic beads and contain all buffers and reagents needed to perform up to 30 ChIP assays. Reagents supplied in the kits are the product of in-house ChIP validation at CST, simplifying your optimization. The kits can be utilized with any ChIP-validated antibody to detect endogenous levels of protein-DNA interactions and histone modifications in mammalian cells. Protein G agarose and Protein G magnetic beads are also available as separate products.

Major Advantages of MNase Enzymatic Digestion Compared to Sonication:

Enzymatic digestion is milder than sonication and better preserves the integrity of the chromatin and antibody epitopes, which means increased IP efficiency.
Increased IP efficiency of target proteins means enhanced detection of protein-bound DNA loci.
Enhanced detection of target DNA loci means less input chromatin required, saving valuable time and reagents.
Enzymatic digestion provides greater sensitivity with lower background, which is essential when detecting DNA bound transcription factors and cofactors.

SimpleChIP Enzymatic Chromatin IP Kit (Magnetic Beads) #9003

Enzyme-based and sonication-based ChIP kits produce chromatin fragments of a similar size. Chromatin was prepared from 4 x 107 HCT116 human colorectal carcinoma cells according to the protocols included with the SimpleChIP^® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003 or a competitor’s sonication-based ChIP kit. DNA was purified from each chromatin sample and DNA fragment size was determined by electrophoresis on a 1% agarose gel. Both enzymatic digestion with the SimpleChIP^® Kit (lane 1) and sonication with the competitor’s kit (lane 2) produced chromatin fragments ranging from 150 to 700 bp, corresponding to one to five nucleosomes in length.

Example A: PCR primers are specific for the transcriptionally active GAPDH and c-MYC genes and the inactive MYT-1 gene.

Example A: Bar graph comparing Tri-Methyl Histone, Acetyl Methyl Histone, Rbp1, and Normal Rabbit IgG based on CST vs. Competitor.

Example B: PCR primers are specific for known TCF4 binding sites in the CAMK2D and c-MYC genes, and a region of the MYT-1 gene that does not contain a TCF4 binding site.

Example B: Bar graph comparing TCF4, beta-Catenin, and Normal Rabbit IgG based on CST vs. Competitor.

SimpleChIP^® digested chromatin is more conducive to immunoprecipitation than sonicated chromatin. Chromatin immunoprecipitations were performed with 10 μg of cross-linked HCT116 chromatin and the indicated antibodies, using SimpleChIP^® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003 or a competitor’s sonication-based ChIP Kit. The enriched DNA was quantified by qPCR. The amount of immunoprecipitated DNA in each sample is presented as a percent of the total input chromatin. For every target tested, enzyme-digested chromatin showed better enrichment of target DNA loci than did sonicated chromatin.