Flow cytometric analysis of DLD-1 cells (blue) and HeLa cells (green) using β2-microglobulin (D8P1H) Rabbit mAb (PE Conjugate).
This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) 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 β2-microglobulin (D8P1H) Rabbit mAb #12851.
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 antibodies. Protect from light. Do not freeze.
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
β2-microglobulin (D8P1H) Rabbit mAb recognizes endogenous levels of total β2-microglobulin protein.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Asp79 of human β2-microglobulin protein.
β2-microglobulin (B2M) is a principal component of the Major Histocompatibility Complex (MHC) class I molecule, a ternary membrane protein complex that displays fragments derived from proteolyzed cytosolic proteins on the surface of cells for recognition by the surveillance immune system (1,2). As an integral component of the MHC class I complex, β2-microglobulin plays a critically important role in immune system function (3). It has important relevance to cancer biology research; for example, research studies have shown that nearly one-third of diffuse large B cell lymphomas contain mutations that inactivate β2-microglobulin gene function, thereby allowing tumor cells to escape immune detection (4). In addition, β2-microglobulin has been identified as an amyloid preprotein with collagen-binding affinity (5); its accumulation in osteoarthritic lesions of long-term dialysis patients is reportedly a contributing factor to the condition known as amyloid osteoarthropathy (6).
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