Chromatin immunoprecipitation (ChIP) is used to determine the location of DNA binding sites for a particular protein of interest, in order to understand the protein-DNA interactions that occur inside the nucleus of living cells or tissues. ChIP is particularly relevant for epigenetics studies, because DNA-binding proteins play an important role in the regulation of gene expression in healthy cells and organisms and in medically relevant areas such as cancer and immunology.The EpiXplore ChIP kits use magnetic beads and an optimized protocol to make your assay easier- in fact, it can be completed in less than 6 hr. These ChIP kits also include specially formulated buffers which reduce nonspecific DNA pull-down and enhance signal. A DNA purifying resin enables rapid elution of DNA and direct PCR analysis.

● Use the EpiXplore Chromatin Immunoprecipitation Kit: Protein G to identify protein-DNA interactions via

protein G, an immunoglobulin-binding protein.

● Use the EpiXplore Chromatin Immunoprecipitation (ChIP) Assay Kit: Anti-mouse IgG to identify protein-DNA

interactions via anti-mouse beads.This kit has significantly higher affinity for mouse antibodies than protein G,

leading to high efficiency pull-down assays when using mouse primary antibodies.

Figure 1. ChIP assay for epigenetic interactions. DNA-binding proteins and crosslinked DNA are immunoprecipitated using a primary antibody specific for the protein of interest, and magnetic beads for easy separation. The protein-DNA crosslinks are then reversed, to release DNA fragments that are ready for direct PCR.

​

□ Highly specific chromatin immunoprecipitation

​

The EpiXplore ChIP Assay Kit yields very specific results. Chromosomal DNA fragments were prepared and treated according to the protocol, with or without anti-RNA-polymerase II antibody. PCR product was obtained from samples captured in the presence of the anti-RNA-polymerase II antibody (Figure 2, lanes 3-4) but not from samples captured in the absence of the anti-RNA-polymerase II antibody (Figure 2, lanes 7-8).Figure 2. Highly specific DNA isolation with the anti-mouse IgG ChIP Assay Kit. Chromosomal DNA fragments were prepared from 5 x 10^6 HEK 293 cells according to the protocol, and incubated ± an anti- RNA Polymerase II antibody. The samples underwent chromosomal capture using anti-mouse IgG magnetic beads and the captured chromosomal DNA was used as a template to amplify a fragment of the GAPDH gene using 18-30 PCR cycles.PCR products were analyzed by DNA-agarose gel electrophoresis. Lanes 1-4, samples treated with anti-RNA Polymerase II antibody. Lanes 5-8, samples treated without anti-RNA II Polymerase Antibody. Lanes 1 and 5: 18 PCR cycles. Lanes 2 and 6: 22 PCR cycles. Lanes 3 and 7: 26 PCR cycles. Lanes 4 and 8: 30 PCR cycles. E, empty lane. M1: 1 kb marker. M2: 100 bp marker.

​

□ Perform ChIP using just a few cells

​

It is possible to perform a successful chromatin immunoprecipitation assay from as few as 1.5 x 105 cells with the EpiXplore ChIP Assay Kit (Figure 3).Figure 3. Perform ChIP assays on small samples. Chromosomal DNA fragments were prepared from 1.5 x 10^5, 3 x 10^5, or 1 x 10^6 HEK 293 cells according to the protocol, and incubated with an antibody against RNA Polymerase II. The samples underwent chromosomal capture using anti-mouse IgG magnetic beads and the captured chromosomal DNA was used as a template to amplify a fragment of the GAPDH gene. PCR products were analyzed by DNA-agarose gel electrophoresis. Lane 1: 1 x 10^⁶ cells. Lane 2: 3 x 10⁶ cells. Lane 3: 1 x 10^⁵ cells. Lane 4: chromatin alone. Lane 5: no template control. M: 1 kb marker.