Pierce Magnetic ChIP Kit

The Thermo Scientific Pierce Magnetic ChIP Kit provides a convenient method for efficient isolation of chromatin-bound DNA by immunoprecipitation (chromatin IP) for subsequent quantitation by PCR.

The Pierce Magnetic ChIP Kit contains sufficient reagents to perform 30 chromatin immunoprecipitation (ChIP) assays with appropriate controls using an optimized protocol. The kit includes reagents for cell lysis, capture of protein-DNA complexes, reversal of crosslinking, and DNA isolation. The blocked Pierce Protein A/G Magnetic Beads provide high binding capacity, low non-specific background, and work with many antibody species. These beads can be used manually with a magnetic stand or with automated platforms, such as the Thermo Scientific KingFisher Instruments. ChIP-validated and quality-guaranteed antibodies are also available for use with the Pierce Magnetic ChIP Kit.

Highlights:

• Rapid – obtain purified DNA that is ready for quantitative PCR in about 8 hours
• Efficient and reproducible – micrococcal nuclease digestion and nuclear lysis are highly optimized
• Sensitive – obtain results with as few as 10,000 cells (1 x 10^4)
• Low background – Pierce Protein A/G Magnetic Beads are blocked in a non-DNA-containing reagent to minimize background
• Complete – optimized positive control reagents are included: RNA polymerase II antibody and GAPDH promoter PCR primers

Includes:

Kit contains reagents for chromatin preparation, IP, DNA purification, and positive controls (antibody and primers)

Requires:

In vivo crosslinker (such as formaldehyde), micro-tip sonicator (such as Misonix* Sonicator 3000), ChIP qualified antibody of choice, PCR primers for DNA sequence of interest, PCR master mix (containing a dye such as SYBR* Green, if qPCR is desired), and a qPCR instrument

Applications:

• Determine sites of specific protein-DNA interactions on genomic DNA by PCR or ChIP-Seq
• Monitor the effects of histone modifications or chemical agents on DNA binding

Product Details:

A successful ChIP assay requires a number of critical steps (crosslinking, chromatin preparation, immunoprecipitation) prior to detecting the target genomic DNA. Individually, each step in the ChIP protocol can be time-consuming to optimize. The Thermo Scientific Pierce Magnetic ChIP Kit simplifies the ChIP process, enabling accurate and reproducible results.

Protein-DNA complexes are first crosslinked in vivo with formaldehyde. The kit contains reagents to lyse cells and extract and solubilize the crosslinked complexes. The complexes are then incubated with a specific antibody and isolated using Pierce Protein A/G Magnetic Beads. After reversing crosslinks and digesting protein, the resulting DNA fragments are purified and are then ready for standard or quantitative PCR.

The Thermo Scientific Pierce Magnetic ChIP Kit provides greater fold enrichment than other kits. LNcaP prostate carcinoma cells were cultured in RPMI-1640 containing 10% FBS for 24 hours. Half of the cultures plated were treated for 16 hours with 5μM camptothecin, a drug that inhibits DNA topoisomerase I. Crosslinking was achieved using a final concentration of 1% formaldehyde in the media for 10 minutes. Using kits from five different suppliers†, ChIP assays were performed according to the manufacturers’ protocols to determine binding of p53 to a 1.5-kb region of the CDKN1A (p21) promoter. Quantitative real-time PCR data was obtained with a Bio-Rad iQ5* Thermocycler. The Pierce Magnetic ChIP Kit has been optimized to isolate even large DNA fragments. † Products compared: Thermo Scientific Pierce Magnetic ChIP Kit, Part No. 26157 Millipore Magna ChIP* A/G, Part No. 17-10085 Life Technologies MAGnify* Chromatin Immunoprecipitation System, Part No. 49-2024 Cell Signaling Technology SimpleChIP* Plus Enzymatic, Part No. 9005 Active Motif ChIP-IT* Express Enzymatic, Part No. 103295

General References:

1. Dey, B, et al. (2012). DNA-protein interactions: methods for detection and analysis. Mol Cell Biochem. 365(1-2):279-99
2. Zeng, P.Y., et al. (2006). In vivo dual cross-linking for identification of indirect DNA-associated proteins by chromatin immunoprecipitation. Biotechniques 41:694-8.
3. Lee, D.Y., et al. (2005). Role of protein methylation in regulation of transcription. Endocr Rev 26(2):147-70.
4. Aparicio, O., et al. (2004). Chromatin immunoprecipitation for determining the association of proteins with specific genomic sequences in vivo. Curr Protoc Cell Biol UNIT 17.7: 17.7.1-17.7.23.
5. Das, P.M., et al. (2004). Chromatin immunoprecipitation assay. Biotechniques 6:961-9.
6. Waldman, T; Kinsler, KW and Vogelstein, B. (1995). p21 is necessary for the p53-mediated G1 arrest in human cancer cells. Cancer Res 55:5187-90.

Related Resources:

Review of methods for detecting Protein-DNA interactions
Review of the ChIP assay method

Related Products:

ChIP-validated Antibodies
Halt Protease and Phosphatase Inhibitors
Subcellular Fractionation Kits