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Glucocorticoid Receptor (D8H2) XP® Rabbit mAb (PE Conjugate) #19560
Flow cytometric analysis of human whole blood using Glucocorticoid Receptor (D8H2) XP® Rabbit mAb (PE Conjugate) (solid line) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (dashed line). Analysis was performed on CD3+ lymphocytes.Learn more about how we get our images
Gallery: Glucocorticoid Receptor (D8H2) XP® Rabbit mAb (PE Conjugate) #19560
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
- 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
- 16% Formaldehyde (methanol free).
- 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.
- 50% methanol.
- 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
- Aliquot 100 μl fresh whole blood per assay tube.
- OPTIONAL: Place tubes in rack in 37°C water bath for short-term treatments with ligands, inhibitors, drugs, etc.
- Add 65 μl of 10% formaldehyde to each tube.
- Vortex briefly and let stand for 15 min at room temperature.
- Add 1 ml of 0.1% Triton™ X-100 to each tube.
- Vortex and let stand for 30 min at room temperature.
- Add 1 ml incubation buffer.
- Pellet cells by centrifugation and aspirate supernatant.
- Repeat steps 7 and 8.
- Resuspend cells in ice-cold 50% methanol in PBS (store methanol solution at -20°C until use).
- Incubate at least 10 min on ice.
- 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.
- Add 2–3 ml incubation buffer to each tube and rinse by centrifugation. Repeat.
- Add primary antibodies diluted as recommended on datasheet or product webpage in incubation buffer.
- Incubate for 30–60 min at room temperature.
- Wash by centrifugation in 2–3 ml incubation buffer.
- 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
Glucocorticoid Receptor (D8H2) XP® Rabbit mAb (PE Conjugate) recognizes endogenous levels of total glucocorticoid receptor protein. Based upon sequence alignment, this antibody is predicted to cross-react with all known alternative translation start site generated isoforms of glucocorticoid receptor-α and glucocorticoid receptor-β. This antibody does not cross-react with mineralocorticoid receptor.Species Reactivity: Human, Mouse, Rat, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu378 of human glucocorticoid receptor protein.
This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometric analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated Glucocorticoid Receptor (D8H2) XP® Rabbit mAb #3660.
Glucocorticoid hormones control cellular proliferation, inflammation, and metabolism through their association with the glucocorticoid receptor (GR)/NR3C1, a member of the nuclear hormone receptor superfamily of transcription factors (1). GR is composed of several conserved structural elements, including a carboxy-terminal ligand-binding domain (which also contains residues critical for receptor dimerization and hormone-dependent gene transactivation), a neighboring hinge region containing nuclear localization signals, a central zinc-finger-containing DNA-binding domain, and an amino-terminal variable region that participates in ligand-independent gene transcription. In the absence of hormone, a significant population of GR is localized to the cytoplasm in an inactive form via its association with regulatory chaperone proteins, such as HSP90, HSP70, and FKBP52. On hormone binding, GR is released from the chaperone complex and translocates to the nucleus as a dimer to associate with specific DNA sequences termed glucocorticoid response elements (GREs), thereby enhancing or repressing transcription of specific target genes (2). It was demonstrated that GR-mediated transcriptional activation is modulated by phosphorylation (3-5). Although GR can be basally phosphorylated in the absence of hormone, it becomes hyperphosphorylated upon binding receptor agonists. It has been suggested that hormone-dependent phosphorylation of GR may determine target promoter specificity, cofactor interaction, strength and duration of receptor signaling, receptor stability, and receptor subcellular localization (3). Indeed Ser211 of human GR is phosphorylated to a greater extent in the presence of hormone, and biochemical fractionation studies following hormone treatment indicate that Ser211-phosphorylated GR is found in the nucleus (3). Thus, Ser211 phosphorylation is a biomarker for activated GR in vivo. An added layer of complexity to GR signaling lies in the ability of multiple isoforms to be generated through both alternative splicing and the use of alternative translation intiation start sites, thus increasing the repertoire of functional signaling homo- and heterodimers (6,7).
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.