Cell Signaling Technology
XP Monoclonal Antibody

Product Pathways - Chromatin Regulation / Epigenetics

Ezh2 (D2C9) XP® Rabbit mAb #5246

Applications Reactivity Sensitivity MW (kDa) Isotype
W IP IHC-P IF-IC ChIP H M R Mk Endogenous 98 Rabbit IgG

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IF-IC=Immunofluorescence (Immunocytochemistry)  ChIP=Chromatin IP
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

Specificity / Sensitivity

Ezh2 (D2C9) XP® Rabbit mAb detects endogenous levels of total Ezh2 protein.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Arg354 of human Ezh2 protein.

Western Blotting

Western Blotting

Western blot analysis of extracts from MCF7, Neuro-2a, and COS-7 cell lines using Ezh2 (D2C9) XP® Rabbit mAb.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Ezh2 (D2C9) XP® Rabbit mAb.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human cervical carcinoma using Ezh2 (D2C9) XP® Rabbit mAb.


IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human lymphoma using Ezh2 (D2C9) XP® Rabbit mAb.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded 4T1a mouse syngeneic tumor using Ezh2 (D2C9) XP® Rabbit mAb.

IF-IC

IF-IC

Confocal immunofluorescent analysis of HeLa cells using Ezh2 (D2C9) XP® Rabbit mAb (green) and S6 Ribosomal Protein (54D2) Mouse mAb #2317 (blue). Actin filaments were labeled with DY-554 phalloidin (red).


Chromatin IP

Chromatin IP

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 NCCIT cells and either 5 µl of Ezh2 (D2C9) XP® Rabbit mAb or 2 µl of Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human HoxA1 Intron 1 Primers #7707, SimpleChIP® Human HoxA2 Promoter Primers #5517, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

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).

  1. Seller, W.B. and Loda, M. (2002) Cancer Cell 2, 349-350.
  2. Visser, H.P. et al. (2001) Br. J. Haematol. 112, 950-958.
  3. Chen, H. et al. (1996) Genomics 38, 30-37.
  4. Tonini, T. et al. (2004) Oncogene 23, 4930-4937.
  5. Muller, J. et al. (2002) Cell 111, 197-208.
  6. Kleer, C.G. et al. (2003) Proc Natl. Acad. Sci. USA 100, 11606-11611.

Application References

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Companion Products

For Research Use Only. Not For Use In Diagnostic Procedures.

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