GAL4 Reporter Kit (Glucocorticoid Receptor Pathway)

Catalog #: 60522
Size: 500 reactions
The glucocorticoid signaling pathway plays an important role in development, fluid homeostasis, cognition, immune response and metabolism. Glucocorticoids are a class of steroid hormones that bind to the glucocorticoid receptor, causing it to translocate to the nucleus. Upon translocation, the receptor can regulate the transcription of a large number of genes, including those that regulate glucose metabolism and inflammatory responses.
The Glucocorticoid Receptor Pathway Reporter Kit is designed for monitoring the activity of the glucocorticoid signaling pathway in cultured cells. The kit contains a transfection-ready expression vector for the glucocorticoid receptor ligand binding domain that is fused to the DNA binding domain (DBD) of GAL4 (GAL4 DBD-GR). This fusion construct activates firefly luciferase expression under the control of a multimerized GAL4 upstream activation sequence (UAS). This allows for specific detection of glucocorticoid-induced activation of the glucocorticoid receptor without the need for individual transcriptional targets and with low crossreactivity for other nuclear receptor pathways. The GAL4/UAS reporter is premixed with constitutively expressing Renilla (sea pansy) luciferase vector, which serves as an internal positive control for transfection efficiency.

The kit also includes a non-inducible firefly luciferase vector premixed with constitutivelyexpressing Renilla luciferase vector as a negative control. The non-inducible luciferase vector contains a firefly luciferase gene under the control of a minimal promoter, but without any additional response elements. The negative control is critical for determining pathway-specific effects and background luciferase activity.

Glucocorticoid Receptor, GR, Nuclear receptor subfamily 3 group C member 1, NR3C1, GAL4, GAL4/UAS, Galectin, Cell based assay kit, cell-based assay, reporter assay kit

Instructions for use:
See assay kit datasheet for detailed protocol.
Storage / Stability:
>1 year from date of receipt when stored at -20°C
• Monitor glucocorticoid signaling pathway activity. • Screen activators or inhibitors of the glucocorticoid signaling pathway.
Paguio A, et al. (2010) Improved Dual-Luciferase Reporter Assays for Nuclear Receptors. Curr Chem Genomics. 4: 43-49.

Materials Required but Not Supplied
• Mammalian cell line and appropriate cell culture medium
• 96-well tissue culture plate or 96-well tissue culture treated white clear-bottom assay plate (Corning # 3610)
• Transfection reagent for mammalian cell line. [We use Lipofectamine™ 2000 (Life Technologies #11668027), however, other transfection reagents should work equally well.]
• Opti-MEM I Reduced Serum Medium (Life Technologies #31985-062)
• Dual luciferase assay system: Dual-Glo® Luciferase Assay System (Promega #E2920): This system assays cells directly in growth medium. It can be used with any luminometer. Automated injectors are not required. OR Dual-Luciferase® Reporter Assay System (Promega #E1910): This system requires a cell lysis step. It is ideal for luminometers with automated injectors.
• Luminometer

These vectors are ready for transient transfection. They are NOT SUITABLE for transformation and amplification in bacteria.

Avoid freeze/thaw cycles.
Scientific Category:
Cell-Based Assay Kit
Product Type:
Assay Kit
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