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Alexa Fluor® 647 Mouse Anti-EZH2
Alexa Fluor® 647 Mouse Anti-EZH2

Flow cytometric analysis of EZH2 expression in human Jurkat cells. Cells from the human Jurkat T cell line (ATCC TIB-152) were fixed in BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeabilized with BD Phosflow™ Perm/Wash Buffer I (Cat. No. 557885) and stained with matching concentrations of either Alexa Fluor® 647 Mouse IgG1, κ Isotype Control (Cat. No. 557732/557783; dashed line histogram) or Alexa Fluor® 647 Mouse Anti-EZH2 antibody (Cat. No. 563491; solid line histogram). The fluorescence histograms were derived from gated events with the forward and side light-scatter characteristics of intact Jurkat cells. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.

Alexa Fluor® 647 Mouse Anti-EZH2

Immunofluorescent Staining of EZH2 in HeLa cells. HeLa cervical adenocarcenoma cells (ATCC CCL-2) were fixed with BD Cytofix™ fixation buffer (Cat. No. 554655), permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050), and stained with Alexa Fluor® 647 Mouse anti-EZH2 monoclonal antibody (Cat. No. 563491, pseudo-colored red) at 5 μg/mL. Counter-staining was with DAPI (pseudo-colored blue). The images were captured on a BD Pathway™ 435 Cell Analyzer and merged using BD AttoVision™ Software. 0.1% Triton™-X 100 is also suitable for permeabilization.

Flow cytometric analysis of EZH2 expression in human Jurkat cells. Cells from the human Jurkat T cell line (ATCC TIB-152) were fixed in BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeabilized with BD Phosflow™ Perm/Wash Buffer I (Cat. No. 557885) and stained with matching concentrations of either Alexa Fluor® 647 Mouse IgG1, κ Isotype Control (Cat. No. 557732/557783; dashed line histogram) or Alexa Fluor® 647 Mouse Anti-EZH2 antibody (Cat. No. 563491; solid line histogram). The fluorescence histograms were derived from gated events with the forward and side light-scatter characteristics of intact Jurkat cells. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.

Immunofluorescent Staining of EZH2 in HeLa cells. HeLa cervical adenocarcenoma cells (ATCC CCL-2) were fixed with BD Cytofix™ fixation buffer (Cat. No. 554655), permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050), and stained with Alexa Fluor® 647 Mouse anti-EZH2 monoclonal antibody (Cat. No. 563491, pseudo-colored red) at 5 μg/mL. Counter-staining was with DAPI (pseudo-colored blue). The images were captured on a BD Pathway™ 435 Cell Analyzer and merged using BD AttoVision™ Software. 0.1% Triton™-X 100 is also suitable for permeabilization.

Product Details
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BD Pharmingen™
Enhancer of zeste 2; ENX-1; Histone-lysine N-methyltransferase EZH2; KMT6
Human (QC Testing), Mouse, Rat, Dog, Chicken (Tested in Development)
Mouse IgG1
Human EZH2 recombinant protein aa. 156-256
Intracellular staining (flow cytometry) (Routinely Tested), Bioimaging, Immunofluorescence (Tested During Development)
5 µl
AB_2738239
Aqueous buffered solution containing BSA, protein stabilizer, and ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was removed.

Recommended Assay Procedures

This antibody conjugate is suitable for intracellular staining of human cell lines using BD Cytofix™ Fixation Buffer. BD Phosflow™

   Perm/Wash Buffer I (Cat. No. 557885) and BD Phosflow™ Perm Buffer III (Cat. No.558050) can  be used with this antibody conjugate.

The Bioimaging protocol can be found at http://www.bdbiosciences.com/support/resources/protocols/ceritifed_reagents.jsp.

Product Notices

  1. This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
  2. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  3. The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
  4. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
  5. Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
  6. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  7. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
  8. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  9. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
  10. An isotype control should be used at the same concentration as the antibody of interest.
  11. Triton is a trademark of the Dow Chemical Company.
563491 Rev. 1
Antibody Details
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11/EZH2

The 11/EZH2 monoclonal antibody specifically binds to the methyltransferase, EZH2 (Enhancer of Zeste Homolog 2). EZH2 is a human homologue of Drosophila's Enhancer of zeste gene, an important regulator of homeobox gene expression. The EZH2 protein has a predicted molecular weight of ~85 kDa. EZH2 is a member of the Polycomb group (PcG) of proteins that are essential for the maintenance, but not initiation, of the transcriptionally repressed state of certain developmental genes. PcG proteins are a structurally diverse group of proteins with conserved functions from fly to human cells. PcG family proteins form multimeric complexes that regulate the expression of genes involved in cell cycle, DNA repair and differentiation. Specifically, EZH2 is a core enzymatic component of PRC2 (polycomb repressive complex 2). EZH2 is expressed in some lymph node follicular T cells and B cells. Thymocytes differentially express EZH2 at various stages during T-cell maturation. EZH2 interacts with multiple signaling proteins, including Vav, that are involved in lymphocyte development and activation. It is highly expressed in a variety of tumors including lymphomas as well as breast and prostate cancers. EZH2 is important in the self renewal and proliferation of numerous stem cell types including fetal hematopoietic stem cells, muscle satellite cells, hepatic stem/progenitor cells, neural stem cells, basal cell progenitors in the developing epidermis, embryonic stem cells, and some cancer stem cells.

563491 Rev. 1
Format Details
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Alexa Fluor™ 647
Alexa Fluor™ 647 Dye is part of the BD red family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 653-nm and an emission maximum (Em Max) at 669-nm. Alexa Fluor 647 is designed to be excited by the Red laser (627-640 nm) and detected using an optical filter centered near 520-nm (e.g., a 660/20 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 647
Red 627-640 nm
653 nm
669 nm
563491 Rev.1
Citations & References
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Development References (10)

  1. Ezhkova E, Pasolli HA, Parker JS, et al. Ezh2 orchestrates gene expression for the stepwise differentiation of tissue-specific stem cells. Cell. 2009; 136(6):1122-1135. (Biology). View Reference
  2. Juan AH, Derfoul A, Feng X, et al. Polycomb EZH2 controls self-renewal and safeguards the transcriptional identity of skeletal muscle stem cells. Genes Dev. 2011; 25(8):789-794. (Biology). View Reference
  3. Kikuchi J, Kinoshita I, Shimizu Y, et al. Distinctive expression of the polycomb group proteins Bmi1 polycomb ring finger oncogene and enhancer of zeste homolog 2 in nonsmall cell lung cancers and their clinical and clinicopathologic significance. Cancer. 2010; 116(12):3015-3024. (Clone-specific: Immunohistochemistry, Western blot). View Reference
  4. Mochizuki-Kashio M, Mishima Y, Miyagi S, et al. Dependency on the polycomb gene Ezh2 distinguishes fetal from adult hematopoietic stem cells. Blood. 2011; 118(25):6553-6561. (Biology). View Reference
  5. Raaphorst FM, Otte AP, van Kemenade FJ. Distinct BMI-1 and EZH2 expression patterns in thymocytes and mature T cells suggest a role for Polycomb genes in human T cell differentiation. J Immunol. 2001; 166(10):5925-5934. (Biology). View Reference
  6. Shen X, Liu Y, Hsu YJ, et al. EZH1 mediates methylation on histone H3 lysine 27 and complements EZH2 in maintaining stem cell identity and executing pluripotency. Mol Cell. 2008; 32(4):491-502. (Biology). View Reference
  7. Simon JA, Lange CA. Roles of the EZH2 histone methyltransferase in cancer epigenetics. Mutat Res. 2008; 647(1-2):21-29. (Biology). View Reference
  8. Su IH, Dobenecker MW, Dickinson E, et al. Polycomb group protein ezh2 controls actin polymerization and cell signaling. Cell. 2005; 121(3):425-436. (Biology). View Reference
  9. Wolters T, Vissers KJ, Bangma CH, Schroder FH, van Leenders GJ. The value of EZH2, p27(kip1), BMI-1 and MIB-1 on biopsy specimens with low-risk prostate cancer in selecting men with significant prostate cancer at prostatectomy. BJU Int. 2009; 106(2):280-286. (Clone-specific: Immunohistochemistry). View Reference
  10. van Kemenade FJ, Raaphorst FM, Blokzijl T. Coexpression of BMI-1 and EZH2 polycomb-group proteins is associated with cycling cells and degree of malignancy in B-cell non-Hodgkin lymphoma. 2001; 97(12):3896-3901. (Biology). View Reference
View All (10) View Less
563491 Rev. 1

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Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims.  Comparisons are not made against non-BD technologies, unless otherwise noted.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.