At what wavelength should PJK luciferase assays be read?
Typically, filters are not used for detection of luciferase activity. In fact, most luminometers do not have a built-in option for discrimination of wavelength and instead read over the entire visible spectrum. The reasons: There is no need to filter and a filtration reduces sensitivity.
What different luciferase assays exists from PJK?
Detection of: Firefly (Photinus Pyralis and similar beetle luciferases) = Beetle-Juice, Beetle LongGlow-Juice, Beetle Lysis-Juice Renilla Reniformis and similar luciferases = Renilla-Juice, Renilla Glow-Juice Gaussia Princeps luciferase = Gaussia-Juice, Gaussia Glow-Juice
Can these luciferases be measured dual (in one sample)?
It exists a patent claims this “dual measurement” of Firefly and Renilla luciferases in one tube. So, we don’t offer this system! But we offer a very reliable “dual well” system that means that the sample with two different luciferases can be split in 2 tubes/wells. These luciferases (all combinations of luciferases are possible that way!!!) can be measured by our reagents in separate wells. Here you will see a comparison of our system to the well known “dual” system in one well:
Dual well System: What’s about the pipetting variation?
Typically you could think that the variation in pipetting will make this method to a very unreliable system. But we proved in several experiments that the variation in pipetting won’t aim higher deviations than the well-known “dual System” in one well does. Following you will find a comparison of both systems:
Renilla/Gaussia-Juices: Can I mix the substrate into the buffer and store it then?
No, you shouldn’t do this. The lyophilisated substrate should be stored in the reconstruction buffer at -80°C and mixed with the buffer just before measurement at room temperature. Remaining reagents should not be freeze again because you will loose activity of the substrate in every way. So, to get reliable results you should prepare fresh reagent before every measurement serie.
Renilla/Gaussia-Juices: High background without any enzyme adding? Why is the background level with Renilla or Gaussia Juice above the usual?
Coelenterazin also emits light from enzyme-regardless oxidation. This process called autoluminescence. In association with lysis components for example Triton X-100, NP 40 or Tween 20 the autoluminescence is enhanced. The same effect appears in the presence of bovine serum albumin (BSA) and Dimethylsulfoxid (DMSO). Please make sure that you don’t have any of these components in your assay (in your lysis reagents etc.)!
Beetle-Juices: Can I store the reagent with the ATP and the substrate?
Yes, you can do this very well at -80 °C. But please make sure that you don’t thaw the reagents more than one time. So, please prepare aliquots of suitable sizes for you measurements. After thawing the reagent please don’t freeze them again. The substrate will loose activity and your results are not reliable anymore.
Beetle Juice has normally a light yellow colour. Why is frozen Beetle Juice white and after the thaw yellow again?
The yellow colour is from D-Luciferin. Beetle Juice is mostly made of water and when Beetle Juice becomes frozen the nature of ice can overbalances and it’s appears white. This not affected the functionality of the assay.
Beetle-Juices: Will I need an automatic injection system to perform an assay with Firefly Luciferase with a luminometer?
A luminometer with automatic Injection system is not essential. The assay with Beetle Juice shows constant light signal in the range of fifteen minutes. For high throughput screenings (HTS) is the Beetle Long Glow- Juice with a stable light signal for 5 hours available.
What is the difference between Beetle-Juice and Beetle Lysis-Juice?
Beetle Lysis Juice includes detergent for cell lysis. For this reason an extra cell lyses step is needless. But to guarantee a proper cell lysis you should particular mix the sample with the reagent through several pipett mixing and wait at least 5 minutes before measurement.Important: It is not useful to combine Beetle Lysis-Juice with Renilla-Juice because of the autoluminescence from coelenterazine in presence of detergents.
What is the matter for different variants from D-Luciferin?
D-Luciferin is available in three forms. D-Luciferin free acid, D-Luciferin potassium salt and D-Luciferin sodium salt. The potassium and sodium salt forms are the most popular because they are readily water-soluble. The potassium salt is also the form used in live animal assay. The small size of Luciferin also makes it a poor antigen and immune responses to Luciferin are unlikely. Luciferin is able to pass the blood brain barrier, the blood placenta barrier and the blood testis barrier, toxicity appears low. L-Luciferin is an enantiomer of D-Luciferin but only the D-enantiomer can provide a proper light signal.
ATP Glow-Juice: Adenosintriphosphate detection for cell viability and bacterial contaminations. Must I use a lysis buffer before I perform the ATP-Glow Juice assay?
No, the ATP Glow-Juice includes detergent for cell lysis. For a proper cell lysis please wait at least 5 min before measuring after adding the buffer to the cells.
ATP Glow-Juice: What is the matter of uncommon high background level of the sample?
Already lowest imprint of ATP in pipette tips and reaction vessels interfere with the assay. To avoid the contamination with ATP use ATP-free materials (for example biopur quality) and wear gloves all the times. If ATP-free materials not available, please use at least autoclaving pipette tips and vessels to reduce the contamination. Based on this facts it’s important to define the background for every sample.
Why do the results disagree intense?
The assay reacts on smallest imprecision. A carefully procedure is essential. I don’t need the entire buffer. How can I store the rest?The ATP-Glow Juice can store with D-Luciferin but without luciferase at -20°C for the least 30 days. Luciferase has be added to this mixture at room temperature just before measuring in the ratio of 1: 1000 (1µl Luciferase in 1 ml buffer)
What is the matter for different variants from Coelenterazine?
Coelenterazine native is the natural substrate for Renilla and Gaussia luciferases. Benzyl Coelenterazine is a synthetic derivative of native coelenterazine. The luminescence intensity of its aequorin complex is 10 times higher than that of the aequorin complex formed from coelenterazine.
It is also more sensitive to Ca2+ and is widely used as a bioluminescent probe for reporter protein such as aequorin and luciferases. All Coelenterazine analogs operate as substrate for Renilla Luciferase with different properties in term of emission wavelength, cell membrane permeability and quantum efficiency. So, these analogs are widely used in invivo assays!