| Antibodies Included | Quantity | Application | Dilution |
|---|---|---|---|
| Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 | 10 immunoprecipitations | ChIP | 1:50 |
| Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb #9733 | 10 immunoprecipitations | ChIP | 1:50 |
| Primers Included | Quantity | Application | Dilution |
|---|---|---|---|
| SimpleChIP® Mouse GAPDH Intron 2 Primers #8986 | 250 PCR reactions | ChIP | 1:10 |
| SimpleChIP® Mouse MYT-1 Promoter Primers #8985 | 250 PCR reactions | ChIP | 1:10 |
| SimpleChIP® Mouse PITX3 Intron 1 Primers #8984 | 250 PCR reactions | ChIP | 1:10 |
ChIP
Product Information
Product Usage Information
Directions for Use:A. Chromatin Immunoprecipitation:ChIP formulated antibodies have been tested and optimized using the SimpleChIP® Enzymatic Chromatin IP Kits (#9002 and #9003). Antibodies should be used at a dilution of 1:50 in a 500 μl ChIP reaction containing 10 to 15 μg of chromatin (4x106 cells). For the SimpleChIP® Enzymatic Chromatin IP protocol, please see the web page for this product at www.cellsignal.com.B. Quantification of DNA by qPCR:1. Label the appropriate number of PCR tubes or PCR plates compatible with the model of real-time PCR machine to be used. PCR reactions should be performed in duplicate and should include a tube with no DNA to control for contamination, and a serial dilution of a 2% total input chromatin DNA (undiluted, 1:5, 1:25, 1:125), which is used to create a standard curve and determine amplification efficiency.2. Add 2 μl of the appropriate ChIP DNA sample to each tube or well of the PCR plate.3. Prepare a master PCR reaction mix as described below. Add enough reagents for two extra reactions to account for loss of volume. Add 18 μl of the master PCR reaction mix to each PCR reaction tube or well of the PCR plate.Reagent Volume for 1 PCR Reaction (20 μl)
Nuclease-free H2O 6 μl
5 μM SimpleChIP® Primers 2 μl
2X SYBR-Green Reaction Mix 10 μl
4. Start the following PCR reaction program:
a. Initial Denaturation 95°C for 3 min
b. Denaturation 95°C for 15 sec
c. Anneal and Extension 65°C for 60 sec
d. Repeat steps b and c for a total of 40 cycles.5. Analyze quantitative PCR results using software provided with the real-time PCR machine.
Storage
Primers are supplied in nuclease-free water at a concentration of 5 μM and should be stored at -20°C.
Specificity / Sensitivity
Species Reactivity:
Mouse
Source / Purification
Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb is produced by immunizing animals with a synthetic peptide corresponding to the amino terminus of histone H3 in which Lys4 is tri-methylated. Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb is produced by immunizing animals with a synthetic peptide corresponding to the amino terminus of histone H3 in which Lys27 is tri-methylated.
Product Description
Background
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.
Species Reactivity
Species reactivity is determined by testing in at least one approved application (e.g., western blot).
Applications Key
ChIP: Chromatin IP
Cross-Reactivity Key
H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected
Trademarks and Patents
Limited Uses
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