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98856S 100 µl (50 tests) $329.00.0
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REACTIVITY SENSITIVITY MW (kDa) Isotype
H M R Endogenous Rabbit IgG
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Flow Cytometry

Flow cytometric analysis of Jurkat cells using RCAS1 (D2B6N) XP® Rabbit mAb (PE Conjugate) (green) compared to Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (red).

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Flow Cytometry General Protocol

If using whole blood, please follow the Flow Cytometry Whole Blood Protocol.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 16% Formaldehyde (methanol free).
  3. 100% methanol.
  4. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Fixation

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Resuspend cells in 0.5–1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
  3. Fix for 10 min at 37°C.
  4. Chill tubes on ice for 1 min.
  5. For extracellular staining with antibodies that do not require permeabilization, proceed to immunostaining (Section D) or store cells in PBS with 0.1% sodium azide at 4°C; for intracellular staining, proceed to permeabilization (Section C).

C. Permeabilization

NOTE: This step is critical for many CST antibodies.

  1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol. Alternatively, remove fix prior to permeabilization by centrifugation and resuspend in 90% methanol as described above.
  2. Incubate 30 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

NOTE: Account for isotype matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies. Count cells using a hemocytometer or alternative method.

  1. Aliquot 0.5–1 x 106 cells into each assay tube (by volume).
  2. Add 2–3 ml incubation buffer to each tube and wash by centrifugation. Repeat.
  3. Resuspend cells in 100 µl of diluted primary antibody (prepared in incubation buffer at the recommended dilution).
  4. Incubate for 1 hr at room temperature.
  5. Wash by centrifugation in 2–3 ml incubation buffer.
  6. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).

E. Optional DNA Dye

  1. Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
  2. Incubate for at least 30 min at room temperature.
  3. Analyze cells in DNA staining solution on flow cytometer.

posted July 2009

revised September 2013

Flow Cytometry Whole Blood Protocol

If using cell lines, please follow the Flow Cytometry General Protocol.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 16% Formaldehyde (methanol free).
  3. Triton™ X-100: To prepare 50 ml of 0.1% Triton™ X-100 add 50 μl Triton™ X-100 to 50 ml 1 X PBS and mix well.
  4. 50% methanol.
  5. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Preparation of Whole Blood (fixation, lysis, and permeabilization) for Immunostaining

  1. Aliquot 100 μl fresh whole blood per assay tube.
  2. OPTIONAL: Place tubes in rack in 37°C water bath for short-term treatments with ligands, inhibitors, drugs, etc.
  3. Add 65 μl of 10% formaldehyde to each tube.
  4. Vortex briefly and let stand for 15 min at room temperature.
  5. Add 1 ml of 0.1% Triton™ X-100 to each tube.
  6. Vortex and let stand for 30 min at room temperature.
  7. Add 1 ml incubation buffer.
  8. Pellet cells by centrifugation and aspirate supernatant.
  9. Repeat steps 7 and 8.
  10. Resuspend cells in ice-cold 50% methanol in PBS (store methanol solution at -20°C until use).
  11. Incubate at least 10 min on ice.
  12. Proceed with staining or store cells at -20°C in 50% methanol.

C. Staining Using Conjugated Primary Antibodies

NOTE: Account for isotype-matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies.

  1. Add 2–3 ml incubation buffer to each tube and rinse by centrifugation. Repeat.
  2. Add primary antibodies diluted as recommended on datasheet or product webpage in incubation buffer.
  3. Incubate for 1 hr at room temperature.
  4. Wash by centrifugation in 2–3 ml incubation buffer.
  5. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer.

Reference: Chow S, Hedley D, Grom P, Magari R, Jacobberger JW, Shankey TV (2005) Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations. Cytometry A 67(1), 4–17.

posted November 2008

revised September 2013

Protocol Id: 407

Product Usage Information

Application Dilutions
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.

Specificity / Sensitivity

RCAS1 (D2B6N) XP® Rabbit mAb (PE Conjugate) recognizes endogenous levels of total RCAS1 protein.


Species Reactivity: Human, Mouse, Rat

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly147 of human RCAS1 protein.

Product Description

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.


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).


1.  Watanabe, T. et al. (1998) Mol Cell Biol 18, 442-9.

2.  Rüder, C. et al. (2009) J Clin Invest 119, 2184-203.

3.  Tsuchiya, F. et al. (2001) Biochem Biophys Res Commun 284, 2-10.

4.  Giaginis, C. et al. (2009) Histol Histopathol 24, 761-76.

5.  Matsushima, T. et al. (2001) Blood 98, 313-21.

6.  Nakashima, M. et al. (1999) Nat Med 5, 938-42.

7.  Reimer, T.A. et al. (2005) BMC Cancer 5, 47.

8.  Wolf, J. et al. (2010) FASEB J 24, 4000-19.

9.  Engelsberg, A. et al. (2003) J Biol Chem 278, 22998-3007.


Entrez-Gene Id 9166
Swiss-Prot Acc. O00559


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.

98856
RCAS1 (D2B6N) XP® Rabbit mAb (PE Conjugate)