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25202
Tox/Tox2 (E6G5O) Rabbit mAb (PE Conjugate)
Antibody Conjugates
Monoclonal Antibody

Tox/Tox2 (E6G5O) Rabbit mAb (PE Conjugate) #25202

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Flow Cytometry Image 1: Tox/Tox2 (E6G5O) Rabbit mAb (PE Conjugate)

Flow cytometric analysis of human peripheral blood mononuclear cells gated on lymphocytes co-stained with CD3 (UCHT1) Mouse mAb (violetFluor™ 450 Conjugate) #61347 using Tox/Tox2 (E6G5O) Rabbit mAb (PE Conjugate) (right) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (left).

Flow Cytometry Image 2: Tox/Tox2 (E6G5O) Rabbit mAb (PE Conjugate)

Flow cytometric analysis of RPMI 8226 cells (blue) and SU-DHL-4 cells (green) using Tox/Tox2 (E6G5O) Rabbit mAb (PE Conjugate) (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (dashed lines).

To Purchase # 25202S
Product # Size Price
25202S
100 µl  (50 tests) $ 345

Supporting Data

REACTIVITY H M R
SENSITIVITY Endogenous
MW (kDa)
Source/Isotype Rabbit IgG

Application Key:

  • W-Western
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • IF-Immunofluorescence
  • F-Flow Cytometry
  • E-P-ELISA-Peptide

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Vir-Virus
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • All-All Species Expected

Product Description

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 Tox/Tox2 (E6G5O) Rabbit mAb #36778.

Product Usage Information

Application Dilution
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.

Protocol

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Flow Cytometry, Methanol Permeabilization Protocol for Directly Conjugated Antibodies

A. Solutions and Reagents

All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below.

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

  1. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100 ml 10X PBS (#12528) to 900 ml water mix.
  2. 4% Formaldehyde, Methanol-Free (#47746)
  3. 100% Methanol (#13604): Chill before use
  4. Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616), or prepare a 0.5% BSA PBS buffer by dissolving 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com for a full listing of cellular dyes validated for use in flow cytometry.

B. Fixation

NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.

NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.

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

NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.

  1. Pellet cells by centrifugation and remove supernatant.
  2. Resuspend cells in approximately 100 µl 4% formaldehyde per 1 million cells. Mix well to dissociate pellet and prevent cross-linking of individual cells.
  3. Fix for 15 min at room temperature (20-25°C).
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS. Proceed to Permeabilization step.
    1. Alternatively, cells may be stored overnight at 4°C in 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. Permeabilize for a minimum of 10 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

  1. Aliquot desired number of cells into tubes or wells. (Generally, 5x105 to 1x106 cells per assay.)
  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 primary antibody, prepared in Antibody Dilution Buffer at a recommended dilution or as determined via titration.
  4. Incubate for 1 hr at room temperature. Protect from light.
  5. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
  6. Resuspend cells in 200-500 µl of 1X PBS and analyze on flow cytometer.

posted July 2009

revised June 2020

Protocol Id: 407

Specificity / Sensitivity

Tox/Tox2 (E6G5O) Rabbit mAb (PE Conjugate) recognizes endogenous levels of total Tox and Tox2 proteins. This antibody does not cross-react with Tox3 or Tox4 proteins. This antibody cross-reacts with an unidentified protein of 50 kDa in some murine cell extracts.

Species Reactivity:

Human, Mouse, Rat

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gln435 of human Tox protein.

Background

Thymocyte selection-associated high mobility group box protein (Tox) is a DNA-binding nuclear factor and member of the evolutionarily conserved high-motility group (HMG)-box superfamily. Tox also defines a small subfamily of proteins that include Tox2, Tox3, and Tox4, all of which are highly conserved in vertebrate species but have unique tissue expression patterns and functions (1,2).

Tox plays a key role in T cell development in the thymus during positive selection (3-5). A study in Tox-deficient mice also revealed a requirement for Tox in CD4 T cell and NK cell lineage development, including NKT cells, FoxP3+ T regulatory T cells, and lymphoid tissue-inducer (LTi) cells (6-8). Although Tox expression is primarily restricted to developing immune cells in normal tissues, Tox is induced by high antigen stimulation during chronic viral infection or cancer, regulating T cell persistence and exhaustion (9-12). Tox has also been shown to be aberrantly expressed in cutaneous T cell lymphomas (13-14).

  1. O'Flaherty, E. and Kaye, J. (2003) BMC Genomics 4, 13.
  2. Aliahmad, P. et al. (2012) Curr Opin Immunol 24, 173-7.
  3. Wilkinson, B. et al. (2002) Nat Immunol 3, 272-80.
  4. Aliahmad, P. et al. (2004) J Exp Med 199, 1089-99.
  5. Chi, T.H. et al. (2002) Nature 418, 195-9.
  6. Aliahmad, P. and Kaye, J. (2008) J Exp Med 205, 245-56.
  7. Aliahmad, P. et al. (2010) Nat Immunol 11, 945-52.
  8. Yun, S. et al. (2011) Immunol Lett 136, 29-36.
  9. Page, N. et al. (2018) Immunity 48, 937-950.e8.
  10. Alfei, F. et al. (2019) Nature 571, 265-269.
  11. Yao, C. et al. (2019) Nat Immunol 20, 890-901.
  12. Wang, X. et al. (2019) J Hepatol pii: S0168-8278(19)30301-0. doi: 10.1016/j.jhep.2019.05.015.
  13. Morimura, S. et al. (2014) Arch Dermatol Res 306, 843-9.
  14. Huang, Y. et al. (2014) Oncotarget 5, 4418-25.

Pathways & Proteins

Explore pathways + proteins related to this product.

Limited Uses

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Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST's products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST's Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

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.
violetFluor is a registered trademark of Tonbo Biosciences.