|88310C||1 Kit (96 assays)||
|Product Includes||Volume (with Count)||Solution Color|
|FastScan™ ELISA Microwell Strip Plate, 96 Well 53257||1 x 96 tests|
|Phospho-Glucocorticoid Receptor (Ser226) Rabbit Capture mAb||1 x 1 ea||Green (Lyophilized)|
|Glucocorticoid Receptor Rabbit HRP-linked mAb||1 x 1 ea||Red (Lyophilized)|
|FastScan™ ELISA Capture Antibody Diluent||1 x 3 ml||Green|
|FastScan™ ELISA HRP Antibody Diluent||1 x 3 ml|
|TMB Substrate 7004||1 x 11 ml|
|STOP Solution 7002||1 x 11 ml|
|Sealing Tape||1 x 1 ea|
|ELISA Wash Buffer (20X) 9801||1 x 25 ml|
|FastScan™ ELISA Cell Extraction Buffer (5X) 69905||1 x 10 ml|
|FastScan™ ELISA Cell Extraction Enhancer Solution (50X) 25243||1 x 1 ml|
|FastScan™ ELISA Kit #88310 Positive Control Type 1||1 x 1 ea|
NOTE: Prepare solutions with deionized/purified water or equivalent.
Prepare only as much reagent as needed on the day of the experiment.
*IMPORTANT: The provided FastScan™ ELISA Cell Extraction Enhancer Solution (50X) may precipitate when stored at 4°C. To dissolve, warm briefly at 37°C and mix gently. The FastScan™ ELISA Cell Extraction Enhancer Solution (50X) can be stored at room temperature to avoid precipitation.
NOTE: The 1X Cell Extraction Buffer contains phosphatase inhibitors. Protease inhibitors should be added to the 1X Cell Extraction Buffer immediately prior to lysing cells. Additional phosphatase inhibitors can also be added (e.g. Protease/Phosphatase Inhibitor Cocktail (100X) #5872, not supplied).
NOTE: A select number of FastScan™ ELISA kits do not contain a positive control, please refer to Product Includes table on the datasheet for a list of included reagents. Should you need support on how to generate a positive control for those kits, contact CST technical support at [email protected].
For adherent cells
For suspension cells
NOTE: Equilibrate all materials and prepared reagents to room temperature prior to running the assay.
*NOTE: Certain FastScan™ ELISA Kits may require additional washes at this step. Any requirements for additional washes will be specifically noted in the product “Description” of the kit’s datasheet.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted May 2018
revised November 2019
Protocol Id: 1924
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 Ser226 by JNK enhances nuclear export after ligand depletion (6,7). Phosphorylation of various serine residues, including Ser226 also affect GR binding to different target genes, contributing to an additional layer of transcriptional regulation (8). Ser226 phosphorylation has also been linked to depression disorders as well as inflammation (9-11).
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