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

COMPONENTS:

See assay kit datasheet for detailed protocol.

>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.

Cell-Based Assay Kit

Assay Kit