How does firefly luciferase work

Flat bottom plates are a must for this assay type do not use round bottom well plates. They were especially designed for optical measurements and cell culture applications.

More information about the flat bottom plate or F-bottom plate can be found at the well plate site. This website also has a more comprehensive list of the different well plate bottoms and what they are designed for. Clear well plates allow you to see your lysates, but the drawback is that you can get background luminescence from neighboring wells.

The white plates with clear bottoms are a solution to the visibility and background issues, but they can be expensive. Just keep those factors in mind in deciding what route to take. The way to answer this question is to understand what constitutes an assay. For example, with our Luciferase Assay Kits , we have kits ranging from 50 assays to 10, assays.

The protocol is written to accommodate a well plate, but this can just as easily be used in a tube. You can order the luciferin, the ATP, and everything else, and then follow the protocols in order to perform the experiment. If you are shopping for individual products, then we encourage you to consider quality, especially when it comes to your luciferin.

The difference in purity can have an impact. Other considerations when choosing luciferin can be found in this article , which goes in some detail about solubility considerations, assay considerations and more. However, the kits present considerable convenience. When making your luciferin stock solution, there is no weigh out required because your luciferin is already measured. It also provides uniformity in your experimental setup.

Another advantage the kit offers is clarity on the products you need. For example, you may have almost all the individual products you need except for the luciferin and the buffer. You might also be unsure about which buffer to choose or how to make the buffer. The kit spares you from a lot of confusion and additional research on what to buy. However, should you need to purchase accessory products, our team is here to help you sort out what you need.

Note: GoldBio does not sell the reaction buffer individually. It is included in the kit, however. Our 5X Luciferase Lysis Buffer is a lysis buffer only. The kit components will vary based on which kit you choose.

Step 1: Choose your luciferase reporter gene firefly luciferase or renilla luciferase, etc. Step 2: Clone your reporter into your plasmid. Step 4: After an incubation period of hours, remove your media and lyse your cells. Step 5: Add the buffer containing luciferin to the lysate. The light from this reaction can be measured with the luminometer. These are the general steps you can expect to follow. There are different types of luciferase assays to choose from. There are flash types and glowing assays.

You could also perform a single assay or a dual luciferase assay in rare cases, even a triple. You might be wondering how to choose. This is all going to depend on what you need for your experiment. The flash type luciferase assay, which is the most common assay type, means that upon adding your substrate, the reaction is going to happen very fast. You have a very limited amount of time to add the substrate and measure the light emission.

It would be impossible in that setup to get an accurate reading. Another option, when running your experiment with a well plate is to pipette substrate into a single well, get a reading, and then move on to your next well — 96 times. As mentioned earlier in this article under the equipment section, some luminometers that measure the light emission from this reaction have been designed with injectors that automate the process of adding substrate, making it immediate and consistent. This ultimately solves the problem you face when working with a lot of samples in a short period of time.

But luminometers with injectors require more substrate for an experiment. The reason is because some substrate is always lost, and it prevents the potential for running out. The benefit of a flash type luciferase assay is that it produces very sensitive results. If this is extremely important for your experiment, and you have the equipment to carry it out, this is the type of luciferase assay you want to choose.

The glowing luciferase assay is an alternative that buys you time. Brasier, A. ScienceDirect; Academic Press. Carter, M. Shieh eds. Cheng, Z. Current Chemical Genomics, 4, 84— Gong, D. Journal of Biological Chemistry, 8 , — Hampf, M. A protocol for combined Photinus and Renilla luciferase quantification compatible with protein assays. Analytical Biochemistry, 1 , 94— He, M. Matta, H. Development and characterization of a novel luciferase based cytotoxicity assay.

Scientific Reports, 8. Min, J. Journal of Clinical Medicine, 9 8 , Nakajima, Y. Multicolor luciferase assay system: one-step monitoring of multiple gene expressions with a single substrate.

BioTechniques, 38 6 , — Smale, S. Luciferase assay. Cold Spring Harbor Protocols, 5 , pdb. What is a Luciferase Reporter Assay? We describe a novel bioluminescence reporter method for the simultaneous monitoring of two genes expressing engineered firefly luciferase variants that emit readily distinguishable green and red light signals.

The key feature is the selectivity of the enzymes for two luciferin substrates that determine each emission color. To validate our method, we performed a complex promoter transactivation experiment side-by-side with the Dual-Luciferase Reporter protocol and obtained essentially identical results.

Additional comparative experiments demonstrated that our assay system provided improvements in background, cell normalization, and detectability compared to representative available methods. With access to a luminometer equipped with two optical filters, this method is an excellent choice for genetic reporter assays that can be performed with a single reagent solution.

Effective methods for monitoring gene expression and regulation based on bioluminescence BL — the emission of light by living organisms — are well known 1 , 2 , 3. The expression of a gene of interest is reported on with high sensitivity and over a wide dynamic range by the emission of light from a variety of engineered luciferase Luc genes from beetles and marine organisms that are expressed downstream of the target gene after exogenous addition of their respective luciferin substrates.

While reporter assays are typically conducted on lysates from cultured human cells, BL detection also enables in vivo cell and animal imaging and biosensor applications as recently reviewed 1 , 2 , 3. When performing reporter assays, especially when using transiently transfected cells, it is essential to normalize the results because controls are needed to account for cell number, viability, and transfection efficiency 4.

Normalization has been achieved in dual-reporter assays that monitor control, e. We have developed a new dual-color assay format called DART D ual A nalyte R eporter with T wo firefly luciferase substrates that combines several advantages of the current method types, while minimizing the drawbacks of each.

A key feature of the two homologous enzyme — two substrate DART method is the selectivity of the Lucs for the substrates that produce very well separated emission spectra. Chemical structures of the benzothiophene BtLH 2 and firefly LH 2 luciferins responsible for each emission color are shown. The lysates were assayed c individually or d mixed together. With this high degree of signal separation Fig. Lysates of equivalent cell count were prepared and mixtures containing both enzymes were assayed with DSM using a microplate luminometer and a single injector.

The simultaneously monitored green and red signals could be measured at levels corresponding to 0. Therefore, optimal results will be obtained using the red signal as the genetic reporter and the green signal as the normalization control.

While negligible overlap of the red signals was detected measured through the green filter with either method Fig. This is because the spectral overlap is approximately equal to the standard deviation of the signal intensity measurements when equal amounts of the Lucs are present. In practice, using PLG3 as the normalization control at a conservative 5 times the background, red signals can be measured over 3 orders of magnitude without increasing the green signal above the error of the measurement.

By increasing the amount of the control plasmid, the dynamic range would remain approximately the same, but higher levels of red signal could be measured as necessary. We also examined the relative intensities of the BL signals emitted by the Lucs used in each method and determined that the intensities of the red- and green-emitting DART Lucs were fold and fold greater than those of the corresponding click beetle enzymes Fig.

These results likely reflect inherent lower specific activity and possibly reduced expression of the click beetle Lucs. This increased to 3. In Fig. In contrast, the data Fig. The novel DART format, which features two substrates and two highly similar firefly Luc enzymes, provides a straightforward and robust method for reporter assays of cell lysates.

The impressive signal separation eliminates the need to do extensive controls and calculations to manage poor signal overlap. The low cost is mainly attributable to the use of DSM, a simple single assay solution that does not contain expensive additives.

The dynamic range of the method spans at least 3 orders of magnitude with exceptionally low background noise. The DART method, however, does require the use of filters and the BtLH 2 substrate is not yet commercially available, but can be obtained from the authors. We are presently investigating the feasibility of extending the principle of the DART method to in vivo imaging applications. Benzothiophene luciferin was prepared as previously described 14 and is available upon request.

The equipment used to make these measurements has been previously described Data were collected from 0. The next day growth media were removed; the cells were washed with PBS, and lysed in 0.

Bioluminescence was initiated by automatic injection of 0. Data shown in Fig. The next day cells were co-transfected in duplicate with plasmids expressing PLR1 reporter under control of the promoter of interest and PLG3 normalization control under control of a CMV or TK promoter. The transfection mix included ng total DNA optimized amounts of each plasmid and empty vector and 1. The next day, growth media were removed; the cells were washed with PBS, and lysed in 0.

Additionally, each reporter-control pair was transfected with pcDNA3. Twenty-four hours after transfection, the cells were washed with PBS and lysed in 0. Cevenini, L. Marks 3—17 Ohmiya, Y.