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26498
Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate)
Antibody Conjugates
Monoclonal Antibody

Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) #26498

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Immunofluorescence Image 1: Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate)

Confocal immunofluorescent analysis of HeLa cells (left) and NIH/3T3 cells (right) using Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) (green) and β-Actin (8H10D10) Mouse mAb #3700 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).

Immunofluorescence Image 2: Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate)

Immunofluorescent analysis of HeLa cells using Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) (green). Prior to immunostaining, antibody was challenged with no peptide (left), peptide corresponding to ubiquitylated Histone H2A(K119) (middle), or peptide corresponding to non-ubiquitylated Histone H2A(K119) (right). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).

Immunofluorescence Image 3: Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate)

Immunofluorescent analysis of HeLa cells using Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate). Prior to immunostaining, increasing concentrations of antibody (x-axis) were challenged with no peptide (green), peptide corresponding to ubiquitylated Histone H2A(K119) (blue), or peptide corresponding to non-ubiquitylated Histone H2A(K119) (red). Fluorescence intensities were then measured to determine optimal working concentration. Minimal blocking was observed with the non-ubiquitylated peptide confirming target specificity.

Flow Cytometry Image 1: Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate)

Flow cytometric analysis of HeLa cells (blue) and C2C12 cells (green) using Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 488 Conjugate) #2975 (dashed lines).

To Purchase # 26498S
製品番号 サイズ 価格 在庫
26498S
100 µl  (50 tests)

Supporting Data

REACTIVITY H M R Mk
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
  • 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 Alexa Fluor® 488 fluorescent dye and tested in-house for direct flow cytometric and immunofluorescent analysis in human and mouse cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAb #8240.

Product Usage Information

Application Dilution
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.

Protocol

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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. Methanol, 100%
  3. 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.
  4. 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.
  5. 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 multi-well plates, chamber slides or on coverslips.

  1. Aspirate liquid, then cover cells to a depth of 2–3 mm with ice-cold 100% methanol.
  2. Allow cells to fix for 15 minutes at -20°C.
  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. Block specimen in Blocking Buffer for 60 minutes.
  2. While blocking, prepare primary antibody by diluting as indicated on product webpage in Antibody Dilution Buffer.
  3. Aspirate blocking solution, apply diluted primary antibody.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 minutes each.
  6. Coverslip slides with Prolong® Gold Antifade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).
  7. For best results, examine specimens immediately using appropriate excitation wavelength. For long-term storage, store slides flat at 4°C protected from light.

posted December 2010

Protocol Id: 221

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

Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) recognizes endogenous levels of histone H2A protein only when ubiquitinated at Lys119. The antibody does not cross-react with other ubiquitinated proteins or free ubiquitin.

Species Reactivity:

Human, Mouse, Rat, Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human histone H2A protein in which Lys119 is mono-ubiquitinated.

Background

The nucleosome, made up of four core histone proteins (H2A, H2B, H3, and H4), is the primary building block of chromatin. Originally thought to function as a static scaffold for DNA packaging, histones have now been shown to be dynamic proteins, undergoing multiple types of posttranslational modifications, including acetylation, phosphorylation, methylation, and ubiquitination (1). Ubiquitin is a conserved 76 amino acid peptide unit that can be covalently linked to many cellular proteins by the ubiquitination process. Three components are involved in this protein-ubiquitin conjugation process. Ubiquitin is first activated by forming a thioester complex with the activation component E1; the activated ubiquitin is subsequently transferred to the ubiquitin-carrier protein E2, then from E2 to ubiquitin ligase E3 for final delivery to the epsilon-NH2 of the target protein lysine residue (2). Histone H2A is mono-ubiquitinated at Lys119 by the Polycomb Repressor Complex 1 (PRC1) and is critical for transcriptional silencing of the developmental HOX genes and X chromosome inactivation (3-6). PRC1 is composed of Bmi1 and RING1A (also RING1 or RNF1), both of which act to enhance the E3 ubiquitin ligase activity of the catalytic subunit RING1B (also RING2 or RNF2) (3,4). Histone H2A is also mono-ubiquitinated at Lys119 at sites of DNA damage. This mono-ubiquitination event requires the PRC1 components Bmi1 and RING1B, in addition to another E3 ubiquitin ligase RNF8, and contributes to subsequent recruitment of the BRCA1 complex, via binding of RAP80/UIMC1 (ubiquitin interactive motif containing 1 protein) (7-10).

  1. Peterson, C.L. and Laniel, M.A. (2004) Curr Biol 14, R546-51.
  2. Liu, F. and Walters, K.J. (2010) Trends Biochem Sci 35, 352-60.
  3. Wang, H. et al. (2004) Nature 431, 873-8.
  4. Cao, R. et al. (2005) Mol Cell 20, 845-54.
  5. de Napoles, M. et al. (2004) Dev Cell 7, 663-76.
  6. Fang, J. et al. (2004) J Biol Chem 279, 52812-5.
  7. Ginjala, V. et al. (2011) Mol Cell Biol 31, 1972-82.
  8. Bergink, S. et al. (2006) Genes Dev 20, 1343-52.
  9. Marteijn, J.A. et al. (2009) J Cell Biol 186, 835-47.
  10. Wu, J. et al. (2009) Mol Cell Biol 29, 849-60.

Pathways & Proteins

Explore pathways + proteins related to this product.

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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.
ProLong is a registered trademark of Life Technologies Corporation.
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 [email protected]
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