QIAGEN Website    Quick Order    Online Seminar    Contact    My Account
Home  >  Products  >  Cignal Reporter Home  >  Reporter Assay  >  TCF/LEF

TCF/LEF Reporter

Luciferase  GFP Lentiviral luciferase Lentiviral GFP
 
Cignal TCF/LEF Reporter (luc) Kit: CCS-018L
The TCF/LEF Reporter kit is designed to monitor the activity of Wnt signal transduction pathways in cultured cells. The kit contains transfection-ready TCF/LEF reporter construct as well as positive and negative controls. The Wnt signaling pathway leads to the dephosphorylation, stabilization, and nuclear translocation of β-catenin. The stabilized β-catenin complexes with the TCF/LEF transcription factors, leading to the activation of Wnt-responsive genes. The TCF/LEF-responsive luciferase construct encodes the firefly luciferase reporter gene under the control of a minimal (m)CMV promoter and tandem repeats of the TCF/LEF transcriptional response element (TRE). We have experimentally optimized the number of response elements as well as the intervening sequence between response elements to maximize the signal to noise ratio. Each reporter is premixed with constitutively expressing Renilla luciferase, which serves as an internal control for normalizing transfection efficiencies and monitoring cell viability.

Using a simple dual-luciferase assay, the TCF/LEF Reporter (luc) can easily and rapidly:

  • Monitor Wnt signaling pathway activity in cells
  • Study chemical compounds or drugs
  • Analyze phenotypes of RNAi or gene over expression

See the complete transcription factor reporter assay list.

 

Kit Components How It Works Manual & Resources Related products
 
10-Pathway Arrays
Component Specification Concentration (total volume)
TCF/LEF Reporter  A mixture of inducible TCF/LEF-responsive firefly luciferase construct and constitutively expressing Renilla luciferase construct (40:1).  (100 ng/µl; 500 µl)
Negative control  A mixture of non-inducible firefly luciferase construct and constitutively expressing Renilla luciferase construct (40:1).  (100 ng/µl; 500 µl)
Positive control  A mixture of constitutively expressing GFP, constitutively expressing firefly luciferase, and constitutively expressing Renilla luciferase constructs (40:1:1).  (100 ng/µl; 250 µl)
 

Kit Components How It Works Manual & Resources Related Products
 
  • Cignal Technology Overview

  • Brief Protocol

The Cignal Reporter Assays (luc) include pre-formulated, transfection-ready reporter, negative control, and positive control. The transcription factor reporter and negative control are transfected and subjected to experimental treatments, in parallel. Dual-luciferase results are calculated for each transfectant. The change in the activity of each signaling pathway is determined by comparing the normalized luciferase activities of the reporter in treated versus untreated transfectants. The identically treated negative control transfectants serve as a specificity control. The positive control serves as a control for transfection efficiency, by monitoring GFP expression, as well as a positive control for both the firefly and Renilla luciferase assays.

  • Performance Data

General performance

Average maximum response rate = 857
Average Z' factor at maximum response rate = 0.87
Average coefficient of variation (CV%) = 7.6%

Excellent signal to noise ratio

Cignal LEF/TCF reporter assay showed upregulation of Wnt signaling activity: 293-H cells were transfected with LEF/TCF reporter, negative control and positive control (for transfection protocol refer our user manual). After 16 hours of transfection, medium was changed to assay medium (Opti-MEM + 0.5% FBS + 0.1mM NEAA + 1mM Sodium pyruvate + 100 U/ml penicillin + 100 g/ml streptomycin). After 24 hours of transfection, cells were treated with either 400 ng/ml of recombinant mouse Wnt3a protein (mWnt3a) for 18 hours, or 14 hours with 10mM LiCl followed by 4 hours with 400 ng/ml of mWnt3a. Dual Luciferase assay was performed 42 hours after transfection, and promoter activity values are expressed as arbitrary units using a Renilla reporter for internal normalization. Experiments were done in triplicates, and the standard deviation is indicated. Cignal LEF/TCF reporter assay measured 857 fold increase in β-catenin activity and, in turn, showed upregulation of Wnt signaling activity by 50 mM LiCl.

Back to Top

 

Kit Components How It Works Manual & Resources Related Products
 
User Manuals Cignal Reporter Assay Kit User Manual (PDF)
White Paper Cignal Reporter Assay Kit: A High Performance Tool for Assessing the Functions of Genes, Biologics and Small Molecule Compounds (PDF)
Web Seminar Attend a live on-line seminar hosted by an Application Scientist about Cignal Reporters
Technical Brochure Cignal Reporter Assay Kits Technical Brochure (PDF)
Power Point Cignal™ Cell-Based Assays for Rapidly Analyzing Signaling Pathway Activity (PDF)
Back to Top
 

Kit Components How It Works Manual & Resources Related Products
 
Pathway reporters Complete list of signaling pathway reporters
Attractene Transfection Achieve high transfection efficiencies and minimal cytotoxicity in your transfections
10-Pathway Arrays Simultaneously measure the activities of ten (10) signaling pathways in a single experiment
shRNA Plasmids Pathway related gene knockdowns - Guaranteed 70% gene knockdown

Back to Top