| Product Includes | Product # | Quantity | Color |
|---|---|---|---|
| FastScan™ ELISA Microwell Strip Plate, 96 Well | 53257 | 96 tests | |
| Phospho-Glucocorticoid Receptor (Ser134) Rabbit Capture mAb | 81633 | 1 ea | Green (Lyophilized) |
| Glucocorticoid Receptor Rabbit HRP-linked mAb | 94554 | 1 ea | Red (Lyophilized) |
| FastScan™ ELISA Capture Antibody Diluent | 16076 | 3 ml | Green |
| FastScan™ ELISA HRP Antibody Diluent | 28120 | 3 ml | |
| TMB Substrate | 7004 | 11 ml | |
| STOP Solution | 7002 | 11 ml | |
| Sealing Tape | 54503 | 1 ea | |
| ELISA Wash Buffer (20X) | 9801 | 25 ml | |
| FastScan™ ELISA Cell Extraction Buffer (5X) | 69905 | 10 ml | |
| FastScan™ ELISA Cell Extraction Enhancer Solution (50X) | 25243 | 1 ml | |
| FastScan™ ELISA Kit #40524 Positive Control Type 1 | 69746 | 1 ea |
*The microwell plate is supplied as 12 8-well modules - Each module is designed to break apart for 8 tests.
Description
The FastScan™ Phospho-Glucocorticoid Receptor (Ser134) ELISA Kit is a sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of
glucocorticoid receptor when phosphorylated at Ser134. To perform the assay, sample is incubated with a capture antibody conjugated with a proprietary tag and a second detection antibody linked to HRP, forming a sandwich with phospho-glucocorticoid receptor (Ser134) in solution. This entire complex is immobilized to the plate via an anti-tag antibody. The wells are then washed to remove unbound material. TMB is then added. The magnitude of observed signal is proportional to the quantity of phospho-glucocorticoid receptor (Ser134).
*Antibodies in this kit are custom formulations specific to kit.
IMPORTANT: This FastScan™ ELISA Kit requires 4 washes at Step 6 of the protocol.
Specificity/Sensitivity
Background
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).
Phosphorylation of GR at Ser134 by p38 MAPK occurs in response to cellular stress and is hormone independent. The phosphorylation event results in stronger association with 14-3-3ζ, which alters chromatin binding and reduces GR transcriptional activity (6).
- Yamamoto, K.R. (1985) Annu. Rev. Genet 19, 209-52.
- Necela, B.M. and Cidlowski, J.A. (2003) Trends Pharmacol. Sci. 24, 58-61.
- Wang, Z. et al. (2002) J. Biol. Chem. 277, 26573-80.
- Rogatsky, I. et al. (1998) J. Biol. Chem. 273, 14315-21.
- Krstic, M. D. et al. (1997) Mol. Cell. Biol. 17, 3947-54.
- Galliher-Beckley, A.J. et al. (2011) Mol Cell Biol 31, 4663-75.
Background References
Cross-Reactivity Key
H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected
Trademarks and Patents
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