Fluidic Analytics – Understand the Machinery of Life
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The luciferase reporter assay is commonly used as a tool to study gene expression at the transcriptional level. It is widely used because it is convenient, relatively inexpensive, and gives quantitative measurements instantaneously.
It has broad applications across various fields of cell and molecular biology – wherever you want to measure or track expression of a cloned gene.
As with many assays and kits, many people who use the luciferase reporter assay don’t take the time to fully understand how it works. But understanding the basics of the techniques you are using is essential for troubleshooting when you hit a problem — and for convincing your thesis examiners that you know what you are talking about!
So let’s dive into the inner workings of the luciferase assay….
The reaction at the heart of the Luciferase Reporter Assay
Luciferases make up a class of oxidative enzymes found in several species that enable the organisms that express them to “bioluminesce,” or emit light. The most famous one of these enzymes is the firefly luciferase. Fireflies are able to emit light via a chemical reaction in which luciferin is converted to oxyluciferin by the luciferase enzyme. Some of the energy released by this reaction is in the form of light.
As an interesting aside… bioluminescence serves a variety of different purposes in nature. Some of these functions include: communication, finding a mating partner, finding food, camouflage, and self-defense. In insects it is thought to play an additional role in oxygen detoxification, a function that presumably evolved more recently. But I digress… back to the assay…
Carrying out the luciferase reporter assay itself
There are several commercially available luciferase reporter assay kits comprising expression vectors that contain the luciferase reporter gene (or a variation of it) and the reagents necessary for the reaction to occur. Just google “luciferase reporter assay” kit, and that should cover it.
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. You then collect the cells, break them open to release all the proteins (including the luciferase), add luciferin and all the necessary cofactors, and measure the enzymatic activity using a luminometer (an instrument that measures light emission from samples and gives you a quantitative reading). Since X is fused to the luciferase reporter gene, the luciferase activity can be directly correlated with the activity of X.
Why you should use the luciferase reporter assay…
This reporter assay can be used to study gene expression as well as other cellular components and events that are involved in gene regulation. Its extreme sensitivity allows quantification of even small changes in transcription, and the availability of results within minutes of completing your experiment makes it even more appealing. All in all, the luciferase reporter assay may be just the thing you need to shed some light on your project from hell!