Bright-Glo™ Luciferase Assay System From Promega

Cell-based assays that use luciferase as a reporter are common in molecular biology. A number of kits are available to measure bioluminescence depending on the specific luciferase in use. The Bright-Glo™ Luciferase Assay System from Promega has been designed specifically for firefly luciferase experiments performed in 96 well plates. Only a single processing step is required to quantify luciferase activity and the reagents are specially formulated to show high sensitivity without a drop in signal. The reagents can be used in a homogeneous manner to lyse cells and quantify luminescence, or a second buffer can be used to lyse, stabilize and store samples.

The kit is available in several sizes to perform 100, 1000 or 10,000 assays calculated on 100 ul reactions in 96 well plates. The kit consists of Bright-Glo™ Luciferase Substrate supplied in a lyophilized form and Bright-Glo™ Luciferase Buffer. To prepare the reconstituted reagent, simply add Buffer to Substrate and mix thoroughly. Once the reagent is prepared, it must either be used within a day or stored at -70ºC and used within a month.

This system requires that all reagents (including cells) be equilibrated to room temperature for best results. For the homogeneous protocol, 100 ul reconstituted reagent is added to the media covering cells in a 96 well plate. The plate is incubated at room temperature for 2 minutes to allow lysis of cells, and the results can then be read within 15 minutes using a luminometer. The non-homogeneous protocol requires the purchase of Glo Lysis buffer from Promega. The media is removed and cells are washed once before the addition of 100 ul Glo Lysis buffer for 5 minutes to lyse the cells. The resulting lysate can be stored at -70ºC, or the results read by adding an equal volume of Bright Glo reagent. Although the non-homogeneous method requires a second buffer to be purchased, the benefit is that luciferase enzyme is stabilized in Glo Lysis buffer, and samples can be left for up to 2 days at room temperature without loss of signal. Another benefit is that cells can be harvested from larger plates using a higher volume of Glo Lysis buffer, and a small aliquot read in a 96 well plate reducing the amount of Bright-Glo™ reagent needed.

I have used this kit on transfected cells with both large and small assays and found it simple and sensitive. The reconstituted reagent retains activity longer than the month recommended by the manufacturer when stored at -70ºC, provided it is aliquoted and subjected to minimal freeze-thaw cycles. However, if you are going to store the reagent for extended periods of time I would recommend using a positive control to ensure the reagent retains activity. The one-step reaction removes the need to wash, lyse and transfer cells, reducing well-to-well variation. However, a drawback of using the homogeneous system for adherent cells is that clear-bottom luminescence plates are expensive. Utilizing the non-homogeneous method may be more cost effective depending on the number of assays performed. Background luciferase activity can also be an issue, depending on the assay. It is important that fist time users incorporate appropriate background controls with each batch of assays as variations in experimental conditions can change results from batch to batch.