Transfection Collection™ : Mouse Notch1/CSL Transient Pack (Notch Signaling Pathway)

Catalog #
79285
$715 *
Size: 100 reactions
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Description

The Notch1/CSL Transient Packis designed to provide the tools necessary for transiently transfecting and monitoring the activity of the Notch signaling pathway in HEK293 cultured cells. The kit contains transfection-ready vectors containing firefly luciferase as a Notch pathway-responsive reporter and constitutively expressing Renilla luciferase as a transfection control.  It also includes the TWO-Step Luciferase detection reagents to detect both luciferase activities and specialized medium for growing and assaying HEK293 cells.

The key to the Notch1/CSL Transient Pack is the expression vector for NOTCH1 that has a deletion of the entire extracellular domain (Notch1DE). Inside the cells, the NOTCH1 DE can be 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 pack 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.

Additionally, the pack includes cell culture medium (BPS Medium 1) that has been optimized for use with HEK293 cells*. BPS Medium 1 includes MEM medium, 10% fetal bovine serum, 1% non-essential amino acids, sodium pyruvate, and 1% Pen/Strep.  Finally, the pack provides the TWO-Step Luciferase (Firefly & Renilla) Assay System. These luciferase reagents provide highly sensitive, stable detection of firefly luciferase activity and Renilla luciferase activity.  The TWO-step luciferase reagents can be used directly in cells in growth medium, and can be detected with any luminometer; automated injectors are not required.          

*Note:  the kit may be used with other cell lines than HEK293, but an alternate cell culture medium may be required for optimal cell growth,

Purchase of this cell line is for research purposes only; commercial use requires a separate license. View the full terms and conditions.

Product Info
Storage and Usage
Citations
Format

Concentration
Both the Reporter and Negative Control Reporter are provided at 60 ng DNA/µl (500 µl each)
UniProt #
P46531
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 to 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/RBPJκ/Suppressor of Hairless/Lag-1) and coactivator Mastermind to turn on transcription of Notch-responsive genes.

References
1. Lu FM et al. (1996) Constitutively active human Notch1 binds to the transcription factor CBF1 and stimulates transcription through a promoter containing a CBF1-responsive element. Proc. Natl. Acad. Sci. USA 93(11): 5663-5667.

2. Kanungo, et al. (2008) The Notch signaling inhibitor DAPT down-regulates cdk5 activity and modulates the distribution of neuronal cytoskeletal proteins. J. Neurochem. 106: 2236.