Luciferase and Beta-galactosidase assays
Cell-based assays are highly efficient screening systems that measure biomolecular changes in cells. These assays are frequently used in molecular biology, biochemical, and pharmaceutical research experiments to study a wide range of biological phenomena. Two cell-based assays that are invaluable to researchers are those that incorporate luciferase and beta-galactosidase. These two technologies are implemented in transfection systems to assess transcriptional activity in transfected cells.
These assays enable scientists to examine biological phenomena such as disease states, cell signaling, responses to stimuli, etc. Other aspects include cell metabolism, how ligands enter the cell, cytotoxicity of substances, actions of receptor molecules, and the localization of foreign particles.
The luciferase protein produces a bioluminescent signal when it comes in contact with the substrate luciferin. Several animals exhibit bioluminescence; various bacteria, Aequorin, the click beetle, and many more are known for their glow. The phenomenon has been associated closely with the firefly, Photinus pyralis, whose particular luciferase is extensively used in molecular biology studies.
Encoded by the lacZ gene in E. coli, beta-galactosidase hydrolyzes lactose into galactose and glucose. Using in vitro systems, scientists replace lactose with o-nitrophenyl-D-galactopyraniside (ONGP), a reaction that releases a yellow color. Researchers then measure the rate at which this yellow color accumulates as a means to determine transfection efficiency.
Scientists tag the luciferase and beta-galactosidase genes to promoter sites of genes whose expression is to be studied. After transfection, host cell lysates are exposed to the appropriate substrates and the lysates show a biochemical activity characteristic of the reporter gene. This means light is emitted when lysates containing luciferase are mixed with its specific substrate (luciferin), and a yellow color appears when lysates containing beta-galactosidase are exposed to ONGP.
Knowing that luciferase and beta-galactosidase are not endogenously expressed across many organisms, cell-based assays that include luciferase and beta-gal can be safely used with different cells belonging to a variety of organisms, both plants and animals.
The luminescent and colorimetric observations function as efficient markers and are easily quantifiable. Due to tagging with the gene of interest, only those cells which are successfully transfected will exhibit these property changes. A measurement of luminescence or yellow color corresponds with the amount of expression of the introduced plasmid.
The luciferase reaction can be measured with the help of a luminometer, while beta-galactosidase absorption can be read using a spectrophotometer at 420 nm. These cell-based assays are multipurpose, easy to perform, non-toxic, non-radioactive, sensitive, fast and high throughput cell screening tools. Aside from being used to determine gene expression in transfection systems, they are employed extensively in drug discovery and for in-depth studies of cellular events.
Luciferase sensitivity enables it to be measured in amounts as low as 10 molecules per cell. The firefly luciferase is naturally translated as a mature enzyme and does not require any modifications, so it starts functioning as soon as it is made. With the growing popularity of cell-based assays, laboratory product manufacturers have come up with an impressive line of cell-based assay kits and reagents.