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).
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).
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 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).
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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.
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.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
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.
posted December 2010
Protocol Id: 221
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.
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.
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.
NOTE: Count cells using a hemocytometer or alternative method.
posted July 2009
revised June 2020
Protocol Id: 407
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.
Human, Mouse, Rat, Monkey
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.
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).
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