Buy Three, Get the Fourth Free! | Start Saving >>
14298
T-Bet/TBX21 (D6N8B) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate)
Antibody Conjugates

T-Bet/TBX21 (D6N8B) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) #14298

APPLICATIONS

REACTIVITY SENSITIVITY MW (kDa) Isotype
H Endogenous Rabbit IgG
IF-IC

Confocal immunofluorescent analysis of NK-92 (positive, left) and K-562 (negative, right) cells using T-bet/TBX21 XP® (D6N8B) Rabbit mAb (green) and β-Actin (8H10D10) Mouse mAb #3700 (red).

Learn more about how we get our images.
Flow Cytometry

Flow cytometric analysis of human PBMCs using T-Bet/TBX21 (D6N8B) XP® Rabbit mAb (Alexa Fluor® 488) co-stained with anti-CD56 (NK cells) and anti-CD19 (B cells) antibodies. T-bet/TBX21 is clearly expressed on the CD56+ NK cell population (left plot) and absent on the CD19+ B cells (right plot).

Learn more about how we get our images.

Immunofluorescence (Immunocytochemistry)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

  1. 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
  2. Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
  3. Methanol, 100%
  4. Blocking Buffer (1X PBS / 5% normal goat serum (#5425) / 0.3% Triton™ X-100): To prepare 10 ml: add 0.5 ml normal goat serum and 0.5 ml 20X PBS to 9.0 ml dH2O, mix. While stirring, add 30 µl Triton™ X-100.
  5. Antibody Dilution Buffer (1X PBS / 1% BSA / 0.3% Triton X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (9998), mix.
  6. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Cultured Cell Lines (IF-IC)

NOTE: Cells should be grown, treated, fixed and stained directly in multiwell plates, chamber slides or on coverslips.

  1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde in 1X PBS.
    NOTE: Formaldehyde is toxic, use only in fume hood.
  2. Allow cells to fix for 15 minutes at room temperature.
  3. Aspirate fixative, rinse three times in 1X PBS for 5 minutes each.
  4. Proceed with Immunostaining (Section C).

C. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

  1. Methanol Permeabilization Step: Cover cells with ice-cold 100% methanol (use enough to cover completely to a depth of 3–5 mm, DO NOT LET DRY), incubate in methanol for 10 minutes at –20°C, rinse in 1X PBS for 5 minutes.
  2. Block specimen in Blocking Buffer for 60 minutes.
  3. While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
  4. Aspirate blocking solution, apply diluted primary antibody.
  5. Incubate overnight at 4°C.
  6. Rinse three times in 1X PBS for 5 minutes each.
  7. Coverslip slides with Prolong® Gold Antifade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).
  8. For best results examine specimens immediately using appropriate excitation wavelength. For long term storage, store slides flat at 4°C protected from light.

posted November 2006

revised December 2010

Protocol Id: 220

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
Immunofluorescence (Immunocytochemistry) 1:200
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.

T-Bet/TBX21 (D6N8B) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) recognizes endogenous levels of total T-Bet/TBX21 protein.

Species Reactivity:

Human

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly465 of human T-Bet/TBX21 protein.

This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 488 fluorescent dye and tested in-house for direct flow cytometry analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated T-bet/TBX21 (D6N8B) XP® Rabbit mAb #13232.

The T-box gene family consists of transcription factors characterized by a related DNA-binding domain (T-box) of approximately 200 amino acids (1,2). The T-box genes exhibit diverse temporal and spatial patterns in the developing embryo. Studies have demonstrated members of this family play crucial roles during embryogenesis in a wide range of organisms by regulating cell fate decisions to establish the early body plan and to regulate later processes underlying organogenesis (3-5). Mutations in T-box genes are associated with many developmental defects (6). Recent studies also indicate potential roles in cancer by members of T-box family (7-9).

  1. Wilkinson, D.G. et al. (1990) Nature 343, 657-9.
  2. Papaioannou, V.E. and Silver, L.M. (1998) Bioessays 20, 9-19.
  3. Showell, C. et al. (2004) Dev Dyn 229, 201-18.
  4. Papaioannou, V.E. (2001) Int Rev Cytol 207, 1-70.
  5. Hoogaars, W.M. et al. (2007) Cell Mol Life Sci 64, 646-60.
  6. Baldini, A. (2004) Curr Opin Cardiol 19, 201-4.
  7. Abrahams, A. et al. (2010) IUBMB Life 62, 92-102.
  8. Rowley, M. et al. (2004) J Mammary Gland Biol Neoplasia 9, 109-18.
  9. Yang, X.R. et al. (2009) Nat Genet 41, 1176-8.
Entrez-Gene Id
30009
Swiss-Prot Acc.
Q9UL17
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).
This product is provided under an intellectual property license from Life Technologies Corporation. The transfer of this product is conditioned on the buyer using the purchased product solely in research conducted by the buyer, excluding contract research or any fee for service research, and the buyer must not (1) use this product or its components for (a) diagnostic, therapeutic or prophylactic purposes; (b) testing, analysis or screening services, or information in return for compensation on a per-test basis; or (c) manufacturing or quality assurance or quality control, and/or (2) sell or transfer this product or its components for resale, whether or not resold for use in research. For information on purchasing a license to this product for purposes other than as described above, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or outlicensing@lifetech.com.

Upstream / Downstream

pathwayImage

Explore pathways related to this product.

To Purchase # 14298S

View sizes
Product # Size Price
14298S
100 µl (50 tests) $ 341.0