Product Pathways - Chromatin Regulation / Epigenetics
SimpleChIP® Human Bivalent Promoter Assay Kit #8982
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.
The SimpleChIP® Human 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 human cells by chromatin immunoprecipitation (ChIP). The SimpleChIP® Human GAPDH Exon 1 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® Human MYT-1 Exon 1 Primers are provided as a positive control for enrichment of tri-methyl Lys27, as MYT-1 is repressed by polycomb proteins in most cell lines. SimpleChIP® Human GATA6 Promoter Primers are provided as a positive control for enrichment of both marks, as the GATA6 promoter is found to be bivalent in human stem cells (7). 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® Human Bivalent Promoter Assay Kit detects endogenous levels of its respective modified histone protein. SimpleChIP® Human GAPDH Exon 1 Primers contain a mix of PCR primers that are specific for amplification of a 68 base pair region of the human GAPDH gene. SimpleChIP® Human MYT-1 Exon 1 Primers contain a mix of PCR primers that are specific for the amplification of an 80 base pair region of the human MYT-1 gene. SimpleChIP® Human GATA6 Promoter Primers contain a mix of PCR primers that are specific for the amplification of a 199 base pair region of the human GATA6 promoter.
NTERA-2 cells were either untreated (left panel) or treated for 15 days with retinoic acid (RA) to induce differentiation along the neuronal lineage (right panel). Chromatin immunoprecipitations were then performed with cross-linked chromatin from 4 x 106 cells 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® Human GAPDH Exon 1 Primers #5516, SimpleChIP® Human MYT-1 Exon 1 Primers #4493, and SimpleChIP® Human GATA6 Promoter Primers #5550. 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 the loss of tri-methyl histone H3 Lys27 on the GATA6 promoter as it is activated during NTERA-2 cell differentiatiation.
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).
- Peterson, C.L. and Laniel, M.A. (2004) Curr Biol 14, R546-51.
- Kubicek, S. et al. (2006) Ernst Schering Res Found Workshop , 1-27.
- Lin, W. and Dent, S.Y. (2006) Curr Opin Genet Dev 16, 137-42.
- Byrd, K.N. and Shearn, A. (2003) Proc Natl Acad Sci USA 100, 11535-40.
- 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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- 9002 SimpleChIP® Enzymatic Chromatin IP Kit (Agarose Beads)
- 9006 ChIP-Grade Protein G Magnetic Beads
- 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.