Product Pathways - Chromatin Regulation / Epigenetics
SimpleChIP® Mouse Bivalent Promoter Assay Kit #8981
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.
The SimpleChIP® Mouse Bivalent Promoter Assay Kit contains ChIP-formulated antibodies and SimpleChIP® primers for the analysis of tri-methyl histone H3 Lys4 and Lys27 marks on target genes in mouse cells by chromatin immunoprecipitation (ChIP). SimpleChIP® Mouse GAPDH Intron 2 Primers are provided as a positive control for enrichment of tri-methyl Lys4, as GAPDH is a housekeeping gene that is heavily enriched for active histone marks. SimpleChIP® Mouse MYT-1 Promoter Primers are provided as a positive control for enrichment of tri-methyl Lys27 enrichment, as MYT-1 is repressed by polycomb proteins in most cell lines. SimpleChIP® Mouse PITX3 Intron 1 Primers are provided for enrichment of both marks, as PITX3 has been shown to be bivalent in many cell types (6). Antibodies and primers are tested and optimized for parallel use with the SimpleChIP® Enzymatic Chromatin IP Kits #9002 and #9003 and SYBR® Green quantitative real-time PCR. The kit provides enough reagents for 10 ChIP assays per antibody and 250 PCR reactions per primer set.
Specificity / Sensitivity
Each antibody in the SimpleChIP® Mouse Bivalent Promoter Assay Kit detects endogenous levels of its respective modified histone protein. SimpleChIP® Mouse GAPDH Intron 2 Primers contain a mix of PCR primers that are specific for amplification of a 200 base pair region of the mouse GAPDH gene. SimpleChIP® Mouse MYT-1 Promoter Primers contain a mix of PCR primers that are specific for the amplification of a 211 base pair region of the mouse MYT-1 gene. SimpleChIP® Mouse PITX3 Intron 1 Primers contain a mix of PCR primers that are specific for the amplification of a 248 base pair region of the mouse PITX3 gene.
Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 mES cells (left panel) or C2C12 cells (right panel) and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb, Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb, or 2 μl of Normal Rabbit IgG, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Mouse GAPDH Intron 2 Primers #8986, SimpleChIP® Mouse MYT-1 Promoter Primers #8985, SimpleChIP® Mouse PITX3 Intron 1 Primers #8984, mouse DLX1 promoter primers, and mouse HOXD10 intron 1 primers. The amount of immunoprecipitated DNA in each sample is normalized for enrichment of total histone H3 and represented as signal relative to the total amount of input chromatin, which is equivalent to one. Note that the PITX3, DLX1 and HOXD10 promoters are all bivalent in stem cells, while only PITX3 remains bivalent in the differenitated cell line C2C12.
The nucleosome, made up of four core histone proteins (H2A, H2B, H3 and H4), is the primary building block of chromatin. Originally thought to function as a static scaffold for DNA packaging, histones have now been shown to be dynamic proteins, undergoing multiple types of post-translational modifications, including acetylation, phosphorylation, methylation and ubiquitination (1). Histone methylation is a major determinant for the formation of active and inactive regions of the genome and is crucial for the proper programming of the genome during development (2,3). Trithorax proteins catalyze the tri-methylation of histone H3 Lys4, a mark of transcriptional activation, while polycomb proteins establish and maintain tri-methylation of histone H3 Lys27, a mark of transcriptional repression (4,5). Though originally thought to be mutually exclusive, recent studies have shown that in stem cells certain developmental genes and highly conserved non-coding elements contain both of these marks (6-8). These ‘bivalent’ regions of the genome are poised for activation and are thought to hold the key to the vast potential of stem cells. As stem cells differentiate along a given lineage, many bivalent genes become monovalent, either retaining the tri-methyl histone H3 Lys4 mark if activated during differentiation, or the tri-methyl-histone H3 Lys27 mark if repressed. Chromatin immunoprecipitation (ChIP) is a powerful technique that can be used to identify bivalent domains in stem cells and changes in bivalency that occur during differentiation (6-8).
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- Kubicek, S. et al. (2006) Ernst Schering Res Found Workshop , 1-27.
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- Cao, R. et al. (2002) Science 298, 1039-43.
- Bernstein, B.E. et al. (2006) Cell 125, 315-26.
- Pan, G. et al. (2007) Cell Stem Cell 1, 299-312.
- Mikkelsen, T.S. et al. (2007) Nature 448, 553-60.
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- 9007 ChIP-Grade Protein G Agarose Beads
- 7017 6-Tube Magnetic Separation Rack
Rabbit monoclonal antibody is produced under license (granting certain rights including those under U. S. Patent No. 5,675,063) from Epitomics, Inc.
For Research Use Only. Not For Use In Diagnostic Procedures.