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Brachyury (D2Z3J) Rabbit mAb (PE Conjugate)

Brachyury (D2Z3J) Rabbit mAb (PE Conjugate) #72209


H Endogenous Rabbit IgG
Flow Cytometry

Flow cytometric analysis of HeLa cells (blue) and MUG-Chor1 cells (green) using Brachyury (D2Z3J) Rabbit mAb (PE Conjugate) (solid line) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (dashed line).

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Flow Cytometry Protocol for the Detection of Transcription Factors

A. Solutions and Reagents

NOTE: Prepare solutions with RODI (reverse osmosis deionized) 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. Formaldehyde (methanol free).
  3. 2% Formaldehyde (stock formaldehyde diluted in PBS; make fresh on day of experiment).
  4. Triton™ X-100.
  5. Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Fixation

NOTE: Immunostaining of surface antigens should be performed on live cells prior to fixation/permeabilization, as per manufacturer’s requirements.

  1. Adjust cell suspension of freshly isolated cells to 1–2 x 106 cells in 100 μl Incubation Buffer per assay tube.
  2. Add antibodies against surface antigens to the assay tubes as per manufacturers’ recommended volume or concentration and incubate for 30 min on ice.
  3. Add 2 ml of Incubation Buffer and wash by centrifugation.
  4. Aspirate supernatant and resuspend in 500 μl of 2% formaldehyde.
  5. Fix for 15 min at room temperature.
  6. Wash 2X by centrifugation in Incubation Buffer.

C. Permeabilization

  1. Add 1ml of 0.1% Triton™ X-100 (v/v in PBS) to the cell pellet.
  2. Resuspend and let stand for 30 min at room temperature.
  3. Wash 2X by centrifugation in Incubation Buffer.

D. Immunostaining

  1. Resuspend cell pellets in 100 μl of antibody working solution, diluted according to individual antibody datasheet or product webpage in Incubation Buffer.
  2. Incubate for 1 hr at room temperature.
  3. Wash 2X by centrifugation in Incubation Buffer.
  4. If using a fluorochrome-conjugated primary antibody, resuspend cells in 350 μl of Incubation Buffer and analyze on flow cytometer; for unconjugated or biotinylated primary antibodies, proceed to Step 5.
  5. Resuspend cells in fluorochrome-conjugated secondary antibody diluted in Incubation Buffer at the recommended dilution.
  6. Incubate for 30 min at room temperature.
  7. Wash 2X by centrifugation in Incubation Buffer.
  8. Resuspend cells in 350 μl of Incubation Buffer and analyze on flow cytometer.

posted December 2013

Protocol Id: 626

Application Dilutions
Flow Cytometry 1:50

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.

Brachyury (D2Z3J) Rabbit mAb (PE Conjugate) recognizes endogenous levels of total Brachyury protein.

Species Reactivity:


Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro256 of human Brachyury protein.

This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometric analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated Brachyury (D2Z3J) Rabbit mAb #81694.

Brachyury protein, encoded by the T gene, is a transcription factor that is vital for the formation of posterior mesoderm and axial development during vertebrate embryogenesis (1). In the mouse, brachyury is necessary for mesodermal morphogenetic cell movements during gastrulation. Brachyury mutant mice die in utero and display deficient mesoderm formation including an abnormal notochord, missing posterior somites, and a reduced allantois (2). Human brachyury is expressed in the notochord, as well as in chordoma tumors that occur along the spine, making it a good marker for notochord and notochord-derived tumors (3,4). A common polymorphism in the human T gene has also been shown to be associated with development of the multifactorial neural tube defect, spina bifida (5,6).

  1. Edwards, Y.H. et al. (1996) Genome Res 6, 226-33.
  2. Wilson, V. et al. (1995) Development 121, 877-86.
  3. Vujovic, S. et al. (2006) J Pathol 209, 157-65.
  4. Yang, X.R. et al. (2009) Nat Genet 41, 1176-8.
  5. Morrison, K. et al. (1996) Hum Mol Genet 5, 669-74.
  6. Jensen, L.E. et al. (2004) Hum Genet 115, 475-82.
Entrez-Gene Id
Swiss-Prot Acc.
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.

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To Purchase # 72209S

Product Number Size Price
72209S 100 µl (50 tests) $299.00.0
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