Luciferase Assay System From Promega

Genetic reporters such as firefly luciferase are commonly used in science to study gene expression, promoter activity, intracellular signal transduction, mRNA processing, protein folding and protein:protein interactions. I used Promega’s Luciferase Assay System to study BMP4 promoter regulation during development in a Xenopus laevis experimental system. Although this system was developed for reporter quantification in mammalian cells, it includes Cell Culture Lysis Reagent which allows reporter quantification in plant and bacterial cells. I used it for lysis of Xenopus laevis embryos and got good and reproducible results without any background. The Luciferase Assay System includes coenzyme A (CoA) which results in kinetic improvement, allowing increased light intensity and greater enzymatic turnover. The light intensity is stable for at least 1 minute.

The setup of the experiment consists of several steps. The first of them is the preparation of lysis buffer. You can choose from three types of buffers: Luciferase Culture Cell Lysis Reagent (CCLR), which provides efficient lysis within minutes; Reporter Lysis Buffer, which requires a freeze-thaw cycle in order to lyse cells efficiently; and Passive Lysis Buffer, which contains an anti-foam agent. The absence of air bubbles may result in more consistent light detection. The next step is the lysis of the cells. I worked with fresh or frozen Xenopus embryos and had good experience with the Luciferase Culture Cell Lysis Reagent (CCLR) provided with kit. About 100 µl CCLR was sufficient to cover the pellet (5embryos per sample) and ensure complete lysis. Cells are lysed by disrupting the pellet or tissue directly in the buffer by either grinding or pipeting.

This kit contains Luciferase Assay Reagent in lyophilized form; before use, the reagent must be mixed with provided buffer. After that the Luciferase Assay Reagent is ready for use and should be kept at 4°C; however, it is very important to bring the Luciferase Assay Reagent to room temperature prior to use. The next step in performing the assay is to aliquot the required amount of Luciferase Assay Reagent into the tubes (if using a single-tube luminometer) or wells in a plate (if using a multi-well plate compatible luminometer). Usually, 100 µl of Luciferase Assay Reagent is sufficient for the reaction. It is important to determine the linear range for your luminometer type, because luminometers can experience signal saturation at high light intensities. To produce a standard curve of light units versus relative enzyme concentration, just make serial dilutions in 1X lysis buffer, supplemented with 1 mg/ml BSA. The addition of BSA is necessary to ensure that luciferase is not lost from solution due to adsorption.

The light emitting reaction happens when you mix your sample with Luciferase Assay Reagent. The instructions suggest using 20 µl cell lysate per reaction but it should be determine experimentally. In my case, I had very efficient transcription or a very sensitive instrument or perhaps both; I found that as little as 5 or 10 µl cell lysate gave me sufficient readings. Also, although the light-emitting reaction happens seconds after mixing the cell lysate with the Luciferase Assay Reagent, it is possible to run measurement again within the first 30 minutes. Also, if you need to delay light measurement, I have found that it is possible to lyse cells and freeze them in Cell Culture Lysis Reagent (CCLR) at -20°C or -80°C until you are ready to take the measurement.

Different types of luminometers can be used with this system. I used a single-tube luminometer with an injector and followed the instructions in the Luciferase Assay manual for this instrument type. Usually, I prepared number of aluminum foil wrapped 1.5 ml tubes with 100 µl with Luciferase Assay Reagent then added 5 µl of the sample lysate and mixed well. I then placed the open tube into the instrument and took the measurement, and proceeded to the next sample.

As a conclusion, I can say that this assay is easy and relatively quick to perform; it gives very reliable and reproducible results. I highly recommend it.