|REACTIVITY||H M R Mk|
|Flow Cytometry (Fixed/Permeabilized)||1:50|
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
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).
Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.
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 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 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.