GAL4 Reporter Kit (Glucocorticoid Receptor Pathway)
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.
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- 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)
- TWO-step luciferase assay system:
- TWO-Step Luciferase (Firefly & Renilla) Assay (BPS Bioscience #60683): This system
assays cells directly in growth medium. It can be used with any luminometer. Automated
injectors are not required. - Luminometer