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.
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.
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.