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.
Sample Type:Cultured cells
Intended Use:Applications include monitoring glucocorticoid signaling pathway activity and for screening activators or inhibitors of the glucocorticoid signaling pathway.
Test Principle:The Glucocorticoid Receptor Pathway Reporter BioAssay™ 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 constitutively expressing 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.
Kit Components:Reporter (Component A), GAL4/UAS luciferase reporter vector + constitutively expressingRenilla luciferase vector, 1x500ul, (60ng DNA/ul)Negative Control Reporter (Component B), Non-inducible luciferase vector + constitutively expressing Renilla luciferase vector, 1x500ul, (60ng DNA/ul)GAL4 DBD-GR (Component C), Expression vector for ligand binding domain of theglucocorticoid receptor + GAL4 DNA binding domain, 1x250ul, (100ng DNA/ul)Negative Control Expression vector (Component D), Expression vector with GAL4 DNA binding domain only, 1x250ul (100ng DNA/ul)
These vectors are ready for transient transfection. They are NOT SUITABLE for transformationand amplification in bacteria.
Storage and Stability:Store powder at 4°C liquid at -20°C. Store other components at 4°C. Stable for at least 6 months For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
