Flow cytometric analysis of Jurkat cells using RCAS1 (D2B6N) XP® Rabbit mAb (PE Conjugate) (solid line) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (dashed line).
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This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometry analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated RCAS1 (D2B6N) XP® Rabbit mAb #12290.
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.
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
RCAS1 (D2B6N) XP® Rabbit mAb (PE Conjugate) recognizes endogenous levels of total RCAS1 protein.
Human, Mouse, Rat
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly147 of human RCAS1 protein.
Receptor binding cancer antigen expressed on SiSo cells (RCAS1) is also known as estrogen receptor-binding fragment-associated gene 9 (EBAG9). Originally identified as an estrogen-inducible gene (1), RCAS1 was recently found to play a novel role in the adaptive immune response by negatively regulating the cytolytic activity of cytotoxic T lymphocytes (CTLs) (2). RCAS1 is conserved in phylogeny and is ubiquitously expressed in most human tissues and cells (3,4). There is evidence that tissue expression of RCAS1 is increased in a variety of malignancies, including cancers of the gastrointestinal tract, liver, lung, breast, ovary, endometrium, and cervix. Research studies have shown that levels of RCAS1 tissue expression are negatively correlated with the prognosis of patients harboring the aforementioned malignancies (4). It is also noteworthy that research studies have detected elevated levels of RCAS1 in the sera of cancer patients (4). Initial studies indicated that RCAS1 was secreted from cancer cells and functioned as a ligand for a putative receptor expressed on NK cells, as well as T and B lymphocytes, inducing their apoptosis, which enabled cancer cells to evade immune surveillance (5,6). Subsequent studies have identified RCAS1 as a type III transmembrane Golgi protein with the ability to regulate vesicle formation, secretion, and protein glycosylation (2,7-9). Indeed, it has been shown that RCAS1 overexpression negatively regulates the cytolytic function of CTLs by negatively regulating protein trafficking from the trans-Golgi to secretory lysosomes (2). Furthermore, RCAS1 overexpression delays vesicle transport from the ER to Golgi and causes components of the ER quality control and glycosylation machinery to mislocalize. As a consequence, RCAS1 induces the deposition of tumor-associated glycan antigens on the cell surface, which are thought to contribute to tumor pathogenesis through the mediation of adhesion, invasion, and metastasis (8,9).
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