Notch Pathway Reporter kit is designed for monitoring the activity of the Notch signaling pathway in cultured cells. The kit contains a transfection-ready expression vector for mouse NOTCH1 that has a deletion of the entire extracellular domain, leaving the transmembrane and intracellular regions intact (Notch1DE). Inside the cells, the NOTCH1 DE is cleaved by γ-secretase and active NOTCH1 NICD is released into the nucleus. The kit also contains CSL (CBF1/RBP-Jk) luciferase reporter vector, which is a Notch pathway-responsive reporter. This reporter contains the firefly luciferase gene under the control of multimerized CSL responsive elements upstream of a minimal promoter. The CSL (CBF1/RBP-Jk) 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.
This kit contains the expression vector for Mouse Notch1DE. We also offer the Human Notch1DE expression vector (BPS 79503), as well as a Mouse Notch1 NICD expression vector which is constitutively active without processing by γ-secretase (BPS Bioscience #79504).
The Reporter and Negative Control Reporter are provided at 60 ng DNA/µl (500 µl each). The Notch1 Expression Vector and Negative Control Expression Vector are provided at 100 ng DNA/µl (250 µl each).
Background
The Notch signaling pathway controls cell fate decisions in vertebrate and invertebrate
tissues. Notch signaling is triggered through the binding of a transmembrane ligand,
such as Delta like ligand 4 (DLL4) or jagged 1 (JAG1) to a Notch transmembrane
receptor (Notch1, Notch2, Notch3, Notch4) on a neighboring cell. This results in
proteolytic cleavage of the Notch receptor, releasing the constitutively active intracellular
domain of Notch (NICD). NICD translocates to the nucleus and associates with
transcription factors CSL (CBF1, RBPJ, Suppressor of Hairless, Lag-1) and the
coactivator, Mastermind, to turn on transcription of Notch responsive genes.
References
1. Lu FM et al. (1996). Proc. Natl. Acad. Sci. USA 93(11): 5663-5667. 2. Kanungo et al (2008). J.Neurochem. 106 2236.
Storage/Stability
Stable at least 12 months from date of receipt, when stored as directed (-20°C)
Instructions for Use
See data sheet for detailed transfection and assay protocols.
Applications
• Screen for activators or inhibitors of the Notch signaling pathway. • Study effects of RNAi or gene overexpression on the activity of the Notch pathway.
Shipping Temperature
-20°C
Notes
The vectors are suitable for transient transfection. They are NOT suitable for transformation and amplification in bacteria.
Warnings
Avoid freeze/thaw cycles
Disclaimers and Limitations
BPS Bioscience assay kits are validated using the listed components according to our specific protocol. Any deviations to kit components or protocols, such as using different proteins, cell/tissue lysates, or buffers (including using the same component from other commercial sources) will void any warranties on the performance of the assay kit and is not recommended.