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Ezh2 (D2C9) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) #45638
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 (Alexa Fluor® 647 Conjugate) (top) or Ezh2 (D2C9) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) (bottom) and co-stained with an anti-human CD3 antibody.Learn more about how we get our images
Gallery: Ezh2 (D2C9) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) #45638
Flow Cytometry, Methanol Permeabilization Protocol for Direct Conjugates
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
- 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
- 16% Formaldehyde (methanol free).
- 100% methanol.
- Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
- Collect cells by centrifugation and aspirate supernatant.
- Resuspend cells in 0.5-1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
- Fix for 15 min at room temperature.
- Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS.
- Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
- Incubate 30 min on ice.
- Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.
- Aliquot desired number of cells into tubes or wells.
- Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
- Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
- Incubate for 1 hr at room temperature. Protect from light.
- Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
- 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
- Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
- Incubate for at least 5 min at room temperature.
- Analyze cells in DNA staining solution on flow cytometer.
posted July 2009
revised June 2017
Ezh2 (D2C9) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) detects endogenous levels of total Ezh2 protein.Species Reactivity: Human, Mouse, Rat, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Arg354 of human Ezh2 protein.
This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 647 fluorescent dye and tested in-house for direct flow cytometry and immunofluorescent analysis in human cells. The 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).
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. Alexa Fluor is a registered trademark of Life Technologies Corporation. The Alexa Fluor dye conjugates in this product are sold under license from Life Technologies Corporation, for research use only excluding use in combination with DNA microarrays and high content screening (HCS).