About the Pierce Agarose ChIP Kit
Overview of the ChIP assay protocol |
The strength of the ChIP assay is its ability to capture a snapshot of specific protein-DNA interactions as they occur in living cells, and then quantitate the interactions using standard or quantitive PCR. A successful ChIP assays requires a number of critical steps to be carried out (crosslinking, chromatin preparation, immunoprecipitation) prior to detecting the target genomic DNA sequence. Individually, each step in the ChIP assay protocol can be time consuming to optimize. The Pierce Agarose ChIP Assay Kit simplifies the process, making accurate and reproducible results easier to obtain.
To perform ChIP assays with the Pierce Agarose ChIP Kit, protein-DNA complexes are stabilized and then extracted. In vivo crosslinking is traditionally achieved with formaldehyde. Crosslinking performed directly in cells locks-in the protein-DNA complexes, trapping these unstable and sometimes transient interactions. To lyse, extract and solubilize the crosslinked complexes, the ChIP assay kit includes the Thermo Scientific Chromatin Prep Module (also sold separately # 26158). Together, this complete ChIP assay kit provides a simple, reliable and convenient means for isolating chromatin-bound DNA and enriching samples for the proteins of interest with less than 15% contamination from other cellular compartments - without a Dounce homogenizer.
| Micrococcal nuclease digestion is more reproducible than sonication for shearing crosslinked chromatin. HeLa cells were cultured in EMEM containing 10% FBS. Crosslinking was achieved by adding formaldehyde directly to cells in growth media to a final concentration of 1% and incubating for 10 minutes. Nuclei were isolated using the Thermo Scientific Chromatin Prep Module (# 26158) and divided into four equal samples. Samples 1 and 2 were processed using a Branson Sonifier*, and enzyme digestion was performed on samples 3 and 4. Following nuclei lysis, crosslinking was reversed and samples were electrophoresed on a 1.2% agarose gel containing SYBR green for 1 hour. Gels were imaged with a UV light. MNase treatment delivered uniform results while sonication yeilded variable average fragments sizes. |
| The Thermo Scientific Pierce Agarose ChIP Kit has greater specific signal and less background than other kits. A431 lung carcinoma cells were cultured in DMEM containing 10% FBS for 24 hours. Following a 24 hour serum withdrawal, half of the cultures plated were treated with 100ng/ml EGF for 10 minutes. Crosslinking was achieved using a final concentration of 1% formaldehyde in the media for 10 minutes. ChIP assays were performed according to the manufacturers' protocols. Quantitive real-time PCR data was obtained with a Bio-Rad iQ5* Thermocycler, Abgene Absolute SYBR Green Fluorescein QPCR master mix, and primers designed to amplify a region of the human MYC promoter proximal to the transcription start site. The Pierce Agarose ChIP Kit exhibited the highest signal to noise ratio. |
| The Thermo Scientific Pierce Agarose ChIP Kit demonstrates binding of NeuroD, C/EBPα and RNA polymerase II to the proximal cFos promoter. SH-SY5Y wild type cells and a stable clone over-expressing TrkB (SH-SY5Y[TRKB]) were cultured for 3 hours in 0.2% charcoal dextran-treated media. Cells were then treated with 100ng/ml of BDNF for 15 minutes. Chromatin-Immunoprecipitation of the three targets using the appropriate antibodies and the Pierce ChIP Kit reveal increased binding to the cFOS promoter with BDNF treatment. |
| The Thermo Scientific Pierce Agarose ChIP Kit is effective for profiling multiple transcription factors. A431 lung carcinoma cells were cultured in DMEM containing 10% FBS for 24 hours. Following a 24 hour serum withdrawal, half of the cultures plated were treated with 100ng/ml EGF for 10 minutes. Crosslinking was achieved using a final concentration of 1% formaldehyde in the media for 10 minutes. ChIP assays were performed with the Pierce ChIP Kit to determine binding of phosphorylated-STAT3, acetylated-CBP, CBP, trimethyl histone H3, acetylated-histone H3, histone H3, and RNA polymerase II to the proximal MYC promoter. Primary antibody amounts were determined empirically. Quantitive real-time PCR data was obtained with a Bio-Rad iQ5* Thermocycler, Abgene Absolute SYBR Green Fluorescein QPCR master mix, and primers designed to amplify a region of the human MYC promoter proximal to the transcription start site. |
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