Upstream / Downstream

pathwayImage

Explore pathways related to this product.

Antibody Guarantee

CST Antibody Performance Guarantee

LEARN MORE  

To Purchase # 96866S

96866S 100 µl (50 tests) $329.00.0
$0.00

Questions?

Find answers on our FAQs page.

ANSWERS  

PhosphoSitePlus® Resource

  • Additional protein information
  • Analytical tools

LEARN MORE

REACTIVITY SENSITIVITY MW (kDa) Isotype
H M R Mk Endogenous Rabbit IgG
Image
Image
Image
Image

Flow Cytometry

Flow cytometric analysis of human peripheral blood mononuclear cells untreated (left) and treated (right) with anti-human CD3 (10 μg/ml, coated plates) and anti-human CD28 (5 μg/ml, 3 days, 37ºC), using Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) (top) or Ezh2 (D2C9) XP® Rabbit mAb (PE Conjugate) (bottom) and co-stained with an anti-human CD3 antibody.

Learn more about how we get our images
Image
Image
Page

Flow Cytometry, Methanol Permeabilization Protocol for Direct Conjugates

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 16% Formaldehyde (methanol free).
  3. 100% methanol.
  4. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Fixation

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Resuspend cells in 0.5-1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
  3. Fix for 15 min at room temperature.
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS.

C. Permeabilization

  1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
  2. Incubate 30 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

  1. Aliquot desired number of cells into tubes or wells.
  2. Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
  3. Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
  4. Incubate for 1 hr at room temperature. Protect from light.
  5. Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
  6. Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).

E. Optional DNA Dye

  1. Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
  2. Incubate for at least 5 min at room temperature.
  3. Analyze cells in DNA staining solution on flow cytometer.

posted July 2009

revised June 2017

Protocol Id: 407

Product Usage Information

Application Dilutions
Flow Cytometry 1:50

Storage: Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.

Specificity / Sensitivity

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


Species Reactivity: Human, Mouse, Rat, Monkey

Source / Purification

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

Product Description

This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometry analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated Ezh2 (D2C9) XP® Rabbit mAb #5246.


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.  Sellers, W.R. and Loda, M. (2002) Cancer Cell 2, 349-50.

2.  Visser, H.P. et al. (2001) Br J Haematol 112, 950-8.

3.  Chen, H. et al. (1996) Genomics 38, 30-7.

4.  Tonini, T. et al. (2004) Oncogene 23, 4930-7.

5.  Müller, J. et al. (2002) Cell 111, 197-208.

6.  Kleer, C.G. et al. (2003) Proc Natl Acad Sci U S A 100, 11606-11.


Entrez-Gene Id 2146
Swiss-Prot Acc. Q15910


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.

96866
Ezh2 (D2C9) XP® Rabbit mAb (PE Conjugate)