R Recombinant
Recombinant: Superior lot-to-lot consistency, continuous supply, and animal-free manufacturing.
Phospho-Ezh2 (Thr345) (E7T7K) Rabbit mAb #45153
Filter:
- WB
- IP
- ChIP
- C&R
Supporting Data
| REACTIVITY | H M R Mk |
| SENSITIVITY | Endogenous |
| MW (kDa) | 98 |
| Source/Isotype | Rabbit IgG |
Application Key:
- WB-Western Blotting
- IP-Immunoprecipitation
- ChIP-Chromatin Immunoprecipitation
- C&R-CUT & RUN
Species Cross-Reactivity Key:
- H-Human
- M-Mouse
- R-Rat
- Mk-Monkey
Product Information
Product Usage Information
For optimal ChIP and ChIP-seq results, use 10 μL of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. This antibody has been validated using SimpleChIP® Enzymatic Chromatin IP Kits.
The CUT&RUN dilution was determined using CUT&RUN Assay Kit #86652.
The CUT&RUN dilution was determined using CUT&RUN Assay Kit #86652.
| Application | Dilution |
|---|---|
| Western Blotting | 1:1000 |
| Immunoprecipitation | 1:50 |
| Chromatin IP | 1:50 |
| Chromatin IP-seq | 1:50 |
| CUT&RUN | 1:50 |
Storage
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/mL BSA, 50% glycerol, and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
Protocol
Specificity / Sensitivity
Phospho-Ezh2 (Thr345) (E7T7K) Rabbit mAb recognizes endogenous levels of total Ezh2 protein only when phosphorylated at Thr345. This antibody may detect a band of unknown origin around 135 kDa.
Species Reactivity:
Human, Mouse, Rat, Monkey
Source / Purification
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr345 of human Ezh2 protein.
Background
The polycomb group (PcG) proteins are involved in maintaining the silenced state of several developmentally regulated genes and contribute to the maintenance of cell identity, cell cycle regulation, and oncogenesis (1,2). Enhancer of zeste homolog 2 (Ezh2), a member of this large protein family, contains four conserved regions including domain I, domain II, and a cysteine-rich amino acid stretch that precedes the carboxy-terminal SET domain (3). The SET domain has been linked with histone methyltransferase (HMTase) activity. Moreover, mammalian Ezh2 is a member of a histone deacetylase complex that functions in gene silencing, acting at the level of chromatin structure (4). Ezh2 complexes methylate histone H3 at Lys9 and 27 in vitro, which is thought to be involved in targeting transcriptional regulators to specific loci (5). Ezh2 is deregulated in various tumor types, and its role, both as a primary effector and as a mediator of tumorigenesis, has become a subject of increased interest (6).
Ezh2 is phosphorylated on Thr345 by cyclin-dependent kinase 1 (CDK1), which increases targeting of Ezh2 to its target loci and promotes methylation of H3 at Lys27 (7,8). This activated form of Ezh2 may deposit initial H3K27me3 marks onto newly synthesized daughter strands of DNA during replication (9). In addition, phosphorylation of Thr345 affects Ezh2 stability by promoting its ubiquitination and subsequent degradation (9).
Ezh2 is phosphorylated on Thr345 by cyclin-dependent kinase 1 (CDK1), which increases targeting of Ezh2 to its target loci and promotes methylation of H3 at Lys27 (7,8). This activated form of Ezh2 may deposit initial H3K27me3 marks onto newly synthesized daughter strands of DNA during replication (9). In addition, phosphorylation of Thr345 affects Ezh2 stability by promoting its ubiquitination and subsequent degradation (9).
- Sellers, W.R. and Loda, M. (2002) Cancer Cell 2, 349-50.
- Visser, H.P. et al. (2001) Br J Haematol 112, 950-8.
- Chen, H. et al. (1996) Genomics 38, 30-7.
- Tonini, T. et al. (2004) Oncogene 23, 4930-7.
- Müller, J. et al. (2002) Cell 111, 197-208.
- Kleer, C.G. et al. (2003) Proc Natl Acad Sci U S A 100, 11606-11.
- Chen, S. et al. (2010) Nat Cell Biol 12, 1108-14.
- Kaneko, S. et al. (2010) Genes Dev 24, 2615-20.
- Wu, S.C. and Zhang, Y. (2011) J Biol Chem 286, 28511-9.
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For Research Use Only. Not For Use In Diagnostic Procedures.
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