What is luciferase reporter assay?
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 become a reporter assay?
To perform the reporter assay, you clone the regulatory region of your gene-of-interest (X) upstream of the luciferase gene in one of these expression vectors, introduce that resulting vector DNA into cells, and let the cells grow for a period of time to allow transcription and translation to occur.
How does a reporter gene work?
Reporter genes can be used to assay for the expression of a gene of interest that is normally difficult to quantitatively assay. Reporter genes can produce a protein that has little obvious or immediate effect on the cell culture or organism. The mRNA is then translated into protein.
What is a constitutive reporter?
“Constitutive” reporters are used for tracking cells. “Inducible” reporters are used for monitoring biological processes. Radionuclide-based reporters can be used in clinical applications.
How is luciferase signal measured?
Cell debris is removed by microcentrifugation and luciferase activity is measured using a luminometer. Some luminometers directly inject the reagents into the cell lysate. Such automation allows the signal to be measured at a precise time following injection, which can increase the consistency of the results.
What wavelength is luciferase assay?
approximately 550 – 570 nm
The luciferase protein from the firefly beetle (Photinus pyralis) is a monomeric enzyme that catalyzes the oxidation of luciferin while emitting light at approximately 550 – 570 nm.
What do reporter assays do?
Reporter gene assays are typically used to measure the regulatory ability of an unknown DNA-sequence. This is done by linking the unknown promoter sequence to an easily detectable reporter gene whose product can be easily detected and quantifiably measured.
What is a transgenic reporter assay?
Transgenic reporters can be utilized to characterize gene regulatory landscapes in three conceptually different ways. One way is to randomly probe different parts of the genome to detect accumulative activity of enhancers (enhancer-trap).
What is a transcriptional reporter?
Transcriptional reporters can be used to rapidly establish a tentative expression pattern for a gene of interest. Fusing 5′ upstream sequences to GFP can be done in a number of ways and usually presents no technical challenge.
What is firefly luciferase assay?
The Luciferase Assay System is an extremely sensitive and rapid reagent for quantitation of firefly luciferase. Linear results are seen over at least eight orders of magnitude of enzyme concentration, and less than 10–20 moles of luciferase can be measured under optimal conditions.
What is a Bret assay?
BRET, or Bioluminescence Resonance Energy Transfer, is a cell-based assay for studying protein-protein interactions. The distance required for the resonance energy transfer to occur (less than 10nm) correlates well with most biological interactions, making BRET ideal for examining macromolecular interactions.
What is the NFAT reporter – Jurkat cell line?
The NFAT Reporter – Jurkat Cell Line contains a firefly luciferase gene under the control of the NFAT response element stably integrated into Jurkat cells.
What is the concentration of human serum in the NFAT assay?
Blinatumomab was tested in the T Cell Activation Bioassay (NFAT) with Raji (CD19+) target cells in the absence or presence of increasing concentrations of pooled normal human serum (0–100% in the antibody sample), resulting in final assay concentration of human serum (0–33.3%).
How do I perform an overnight TCR/CD3 effector cells (NFAT) assay?
Follow the steps below using aseptic technique in a sterile cell culture hood if performing an overnight assay. 1. Remove one vial of TCR/CD3 Effector Cells (NFAT) from storage at –140°C and transfer to the bench on dry ice. 2. Add 3.2ml of prewarmed (37°C) assay buffer to a 15ml conical tube. 3.
Where can I find the technical literature for T cell activation bioassay (NFAT)?
All technical literature is available at: www.promega.com/protocols/ Visit the web site to verify that you are using the most current version of this Technical Manual. E-mail Promega Technical Services if you have questions on use of this system: [email protected] T Cell Activation Bioassay (NFAT) 1. Description