What is top Flash assay?
The TopFlash assay is a luciferase reporter assay, and it can be used to monitor the concentration of both Wnt and R-spondin in conditioned media. The assay begins when 2 luciferase expressing plasmids (one of inducible and the other of constitutive expression) are transfected into HEK293 cells.
What is TOPFlash?
TOPFlash is a luciferase reporter that contains a minimal fos promoter coupled to Tcf-binding sites upstream of a modified firefly luciferase gene. The dual luciferase system of Promega was used with a luminometer to measure expression levels in light units. UPF1 CRISPR activation.
What is luciferase reporter assay?
A luciferase reporter assay is a test that investigates whether a protein can activate or repress the expression of a target gene using luciferase as a reporter protein (Carter & Shieh, 2015). This bioluminescence directly corresponds with the effect of the protein on expression of the target gene.
How do I activate Wnt signaling path?
To activate Wnt signaling, one can add Wnt protein, either in a purified form or as conditioned medium to cells. Cells producing active Wnt can be obtained from the ATCC (see also the reagents page). Active Wnt protein can be obtained from several companies.
What is Wnt protein?
Wnt proteins are secreted, lipid-modified glycoproteins that allow for communication between cells. They regulate cell growth, function, differentiation, and cell death. Wnt proteins play a central role in bone development, modeling, and remodeling.
What is a luciferase assay?
A luciferase assay is used to determine if a protein can activate or repress the expression of a target gene. Luciferase is an enzyme used for bioluminescence by various organisms in nature, most famously the firefly.
What is the difference between luciferase and luciferin?
One is a luciferin, or a light-producing substance. The other is a luciferase, or an enzyme that catalyzes the reaction. In some cases, the luciferin is a protein known as a photoprotein, and the light-making process requires a charged ion to activate the reaction.
How do I know if my Wnt is signaling?
Most cells respond to Wnt signaling by an increase in the levels of beta-catenin. There are good antibodies to detect beta-catenin either on Western Blots or by staining (Transduction labs).