NovaBright™ Chemiluminescent Reporter Gene Detection Assays
Ultrasensitive Reporter Gene Assays
NovaBright™ reporter gene assays offer chemiluminescent measurement of reporter gene activity that is more sensitive than colorimetric or fluorescent detection. NovaBright™ kits are available for the most common reporter genes, and are optimized to yield consistent results with high sensitivity over a wide dynamic range spanning femtogram to nanogram amounts of reporter.
Reporter Gene Assays
Studying the Regulation of Gene Expression
Reporter gene assays are invaluable for studying the regulation of gene expression by cis-acting factors (gene regulatory elements) or trans-acting factors (transcription factors or exogenous regulators). In these assays, the reporter gene acts as a surrogate for the coding region of the gene under study.
The reporter gene construct contains one or more gene regulatory elements being analyzed, the structural sequence of the reporter gene, and the sequences required for the formation of functional mRNA. Upon introduction of the reporter construct into cells, expression levels of the reporter gene are monitored through a direct assay of the reporter protein’s enzymatic activity.
The sensitivity of each reporter gene assay is a function of several factors including detection method, reporter mRNA and protein turnover, and endogenous (background) levels of the reporter activity. Both protein turnover and levels of endogenous background vary with each reporter protein and the cell line used. Commonly used detection techniques utilize isotopic, colorimetric, fluorometric, or luminescent enzyme substrates and immunoassay-based procedures with isotopic, colorimetric, or chemiluminescent endpoints.
Common Reporter Genes
Beta-Galactosidase is traditionally detected with the colorimetric substrate o-nitrophenyl-β-D-galactopyranoside (ONPG) , and is often used in conjunction with other reporter genes to normalize transfection efficiency. As indicated in the table [web designer link to table], this colorimetric assay is less sensitive compared to many other reporter gene assays. With NovaBright™ 1,2-dioxetane chemiluminescent substrates for beta-galactosidase, the sensitivity is increased dramatically [2,3].
- Learn more about NovaBright™ Beta-galactosidase Assays
Secreted Placental Alkaline Phosphatase
Secreted placental alkaline phosphatase (SEAP) is secreted by cells directly into the culture medium, and can be assayed simply by taking samples of cell culture medium. Secreted reporter proteins enable the nondestructive assay of cell culture medium, preserving cells for additional assays and enabling time-course monitoring of gene expression. SEAP is detected with both colorimetric and chemiluminescent substrates. NovaBright™ chemiluminescent SEAP reporter gene assays exhibit remarkable sensitivity and ease of use.
- Learn more about NovaBright™ SEAP Assays
Luciferase has become increasingly popular as a reporter gene, especially for cotransfection experiments where it is important to normalize transfection efficiency. The NovaBright™ β-galactosidase and firefly luciferase dual enzyme reporter gene assay offers high sensitivity and a simple assay procedure, and enables the user to perform both measurements from a single aliquot of cell extract in the same reaction well or tube, which minimizes experimental error.
- Learn more about NovaBright™ Beta-galactosidase & Firefly Luciferase Assays
Comparison of Reporter Gene Assays
||Detection Method||Detection Limit||Advantages||Disadvantages|
|Chloramphenicol acetyltransferase (CAT)||Isotopic,
|5 x 107 molecules,
1 x 109 molecules
Low dynamic range,
High cost per assay
NovaBright™ beta-galactosidase system,
NovaBright™ SEAP system
|3 x 108 molecules,
6 x 105 molecules,
3 x 103 molecules
Wide dynamic range,
|Human growth hormone||Radioimmunoassay||3 x 108 molecules||Secreted into media||Radioactivity,
High cost per assay,
|Luciferase||NovaBright™ dual beta-galactosidase and firefly luciferase system||103–104 molecules||Assay simplicity,
Wide dynamic range
|Secreted placental alkaline phosphatase||pNPP (color),
NovaBright™ SEAP systems
|1 x 108 molecules,
3 x 104 molecules
|Secreted into media,
Wide dynamic range
- Alam J, Cook JL (1990) Reporter genes: application to the study of mammalian gene transcription. Anal Biochem 188:245–254.
- Bronstein I, Martin CS, Fortin JJ, Olesen CE, Voyta JC (1996) Chemiluminescence: Sensitive detection technology for reporter gene assays. Clin Chem 42:1542–1546.
- Bronstein I, Fortin J, Stanley PE, Stewart GS, Kricka LJ (1994) Chemiluminescent and bioluminescent reporter gene assays. Anal Biochem 219:169–181.