It will mainly depend on the amount of luciferase present in your assay. You always want to have the substrate in excess. We recommend to use anything between 10 and 100 µM substrate in a buffered solution that will be mixed in equal volumes with your cell culture or protein solution.
If you are using the recommended amount of 100uM you can run at least 2x 96-well plates with 1 mg of Coelenterazine. This is also including volume that is used to prime the pumps in a luminometer with injection port.
All of our dried substrates are either vacuum sealed or packaged under Argon atmosphere. The substrate in the unopened vessel is stable for at least 3 years if stored at -80ºC. Once dissolved in NanoFuel Solvent it will be stable for at least one year at -20ºC or below. We can’t guarantee the stability in a solvent of your choice (e.g. Ethanol, Methanol), since the auto-oxidation will depend on the water content of the alcohol and its overall quality.
NanoFuel Solvent was specifically formulated to protect the Coelenterazine compound from oxidation. Since Ethanol/Methanol is very hygroscopic, we recommend to alcohol from an unopened bottle. Anhydrous, 200-proof Ethanol would be optimal. In addition degassing and acidifying the alcohol will also benefit the shelf-life of the substrate.
The photoprotein Aequorin is showing an up to 20-fold higher inital intensity with h-CTZ compared to native Coelenterazine.
h-CTZ is also the preferred substrate for Renilla Luciferase in HTS (high throughput screens) due to its prolonged glow kinetic.
h-CTZ is mainly used for high troughput screens due to its glow kinetic in conjunction with Renilla Luicerase.
For maximum light-output (up to 470%) we recommend to use CTZ-F with Renilla Luciferase.
For maximum stability and shelf-life of the substrate we recommend NanoFuel Solvent (Cat.# 399).
As an alternative you may use Methanol to dissolve h-CTZ.
We recommend using our NanoFuel Solvent (Cat.#399). As an alternative Methanol can be used. Please note: Methanol is very hygroscopic and will have an increased water content over time. Please use fresh Methanol to dissolve the substrate.
If you use NanoFuel Solvent (Cat. #399) to dissolve the substrate, it will be stable for at least one year stored at -20C or below.
Methanol dissolved CTZ400a should have the same shelf-life. Methanol is very hygroscopic and will have an increased water content over time. Please use fresh Methanol to dissolve the substrate.
There are a few mutated versions of Renilla Luciferase (e.g. RLuc8, RLuc3) available that will give you an increase in signal.
You can also change the substrate to Prolume Purple (Cat.#369) which will have a similar emission peak but with an up to 10-fold higher intensity.
Yes. Due to its additional ring structure it is more susceptible to auto-oxidation. Please store at -80°C.
eCTZ-F is showing unique properties in the emission spectrum, peaking at 420 and 480 nm.
In terms of reaction kinetic, eCTZ-F is displaying a prolonged signal with a higher inital intensity using Renilla Luciferase compared to native Coelenterazine.
Prolume developed eCTZ-F for commercial usage. Therefore it is a very new compound to the scientific community. Please contact us if you are interested in a collaboration regarding eCTZ-F.
vCTZ is very difficult to dissolve even in Methanol or Ethanol. In order to reach high concentrations use NanoFuel-V Solvent (Cat. #398).
CTZ-V and its derivatives have a triple carbon bond requiring a special solvent in order to make up higher concentrated solutions.
NanoFuel-Solvent was specifically prepared to preserve the luciferins as long as possible. We only use fresh HPLC grade reagents that were additionally dried and degased to remove any oxidizing compounds. We can’t guarantee the product stability if you used a 5 year old bottle of denatured alcohol to dissolve your CTZ.
