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Luciferase Assays

No. Biotium’s Renilla and Firefly Assay Kits are based on flash luminescence. Samples should be treated and read individually. Alternatively, you may purchase the Steady-Glo Firefly Assay Kit for HTS. We currently do not have a Steady-Glo for Renilla Assays.

The Passive Lysis Buffer (Renilla) is compatible with the Firefly Assay, but the Firefly Lysis Buffer is not compatible with the Renilla Assay.

Luminescence values are relative and depend on the experimental system. For example, luminometers from two different manufacturers each may have 6-log capability, but one may measure a range of 1 to 1,000,000 relative light units (RLU) while the other measures 0.01 to 10,000 RLU. Both instruments can measure a dynamic range of 6-log units, but they read out different arbitrary units.

Biotium’s Renilla and Firefly Assays are flash luminescence assays. The approximate half-lives are 1-2 minutes for Renilla and 10 minutes for Firefly. The Steady-Glo Firefly Assay has a half-life of about 3-5 hrs. However, the signal is about 1/10 of the flash luminescence assays.

No. The buffers are based on different proprietary formulations.

Renilla is often used as an internal control for transfection efficiency while the Firefly luciferase vector is used for reporter activity. They also can be reversed, with Renilla as the reporter and Firefly as the internal control.

Yes, please check the website for catalog numbers and pricing.

Assay sensitivity is dependent on many factors including the promoter strength of each vector, the transfection efficiency of each vector, and the cell type used. In general, Firefly luciferase is more widely used because it has a longer half-life and and better stability than Renilla luciferase.

Some of Biotium’s luciferase assay kits can be used as alternatives to Promega kits. The comparable products are listed below. Click on a link for product information.

Promega assayPromega catalog no.Biotium alternativeBiotium catalog no.Assay typeNotes
Steady-Glo® Assay SystemE2510, E2520, E2550Steady-Luc™ Firefly HTS Assay Kit30028Glow-type
Signal half-life 3h
Signal half-life ~3 hours. Luciferase substrate is added to assay buffer as needed, so signal doesn't drop during kit storage.
Steady-Luc™ Firefly HTS Assay Kit (Lyophilized)30028LGlow-type
Signal half-life 3h
Signal half-life ~3 hours. Lyophilized format for convenient shipping and storage.
Firefly Luciferase Assay SystemE1483, E1500, E1501, E4030, E4530, E4550Firefly Luciferase Assay Kit 2.030085Flash-typeLuciferase substrate is added to assay buffer as needed, so signal doesn't drop during kit storage.
Firefly Luciferase Assay Kit (Lyophilized)30075Flash-typeLyophilized format for convenient shipping and storage.
Renilla Luciferase Assay SystemE2810, E2820Renilla Luciferase Assay Kit 2.030082Flash-typeFeatures Biotium's water-soluble Aquaphile™ Coelenterazine
Dual-Luciferase® Assay System
(Firefly & Renilla)
E1910, E1960, E1980Firefly & Renilla Luciferase Single Tube Assay Kit30081Flash-typeSequentially measure firefly and Renilla luciferase activities in the same sample in one tube.
CellTiter-Glo® Luminescent Cell Viability AssayG7570, G7571, G7572, G7573ATP-Glo™ Cell Viability Assay30020Flash-typeCell Titer Glo® is a glow type assay (5h half-life); ATP-Glo™ is a flash type assay (signal stable for 1 minute)

Most of our products are stable at room temperature for many days, so in all likelihood the product will still work just fine. To be on the safe side, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

One exception that we are aware of is GelGreen™, which is more sensitive to light exposure than most of our other fluorescent dyes. If GelGreen™ is exposed to ambient light for a prolonged period of time (days to weeks), its color will change from dark orange to brick red. If this occurs, the GelGreen will no longer work for gel staining.

 

Bioscience kits
The guaranteed shelf life from date of receipt for bioscience kits is listed on the product information sheet. Some kits have an expiration date printed on the kit box label, this is the guaranteed shelf life date calculated from the day that the product shipped from our facility. Kits often are functional for significantly longer than the guaranteed shelf life. If you have an older kit in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the kit still works for your application before processing a large number of samples or precious samples.

Antibodies and other conjugates
The guaranteed shelf life from date of receipt for antibodies and conjugates is listed on the product information sheet. Antibodies and other conjugates often are functional for significantly longer than the guaranteed shelf life. If you have an older conjugate in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

For lyophilized antibodies, we recommend reconstituting the antibody with glycerol and antimicrobial preservative like sodium azide for the longest shelf life (note that sodium azide is not compatible with HRP-conjugates).

Chemicals, dyes, and gel stains
Biotium guarantees the stability of chemicals, dyes, and gel stains for at least a year from the date you receive the product. However, the majority of these products are highly stable for many years, as long as they are stored as recommended. Storage conditions can be found on the product information sheet or product safety and data sheet, material safety data sheet, and on the product label. Fluorescent compounds should be protected from light for long term storage.

If you have a Biotium compound that has been in storage for longer than one year that you wish to use, we recommend performing a small scale positive control experiment to confirm that the compound still works for your application before processing a large number of samples or precious samples.

Expiration date based on date of manufacture (DOM)
If your institution requires you to document expiration date based on date of manufacture for reagents, please contact techsupport@biotium.com for assistance.

Chemical products with special stability considerations:

Esters

Ester compounds include the following:
• Succinimidyl esters (SE, also known as NHS esters), such as our amine-reactive dyes
• Acetoxymethyl esters (AM esters) such as our membrane-permeable ion indicator dyes
• Diacetate-modified dyes, like ViaFluor™ 405, CFDA, and CFDA-SE cell viability/cell proliferation dyes

Ester dyes are stable in solid form as long as they are protected from light and moisture. Esters are not stable in aqueous solution. Concentrated stock solutions should be prepared in anhydrous DMSO (see Biotium catalog no. 90082). Stock solutions in anhydrous DMSO can be stored desiccated at -20°C for one month or longer. Esters should be diluted in aqueous solution immediately before use. Succinimidyl esters (SE) should be dissolved in a solution that is free of amine-containing compounds like Tris, glycine, or protein, which will react with the SE functional group. AM esters and diacetate compounds should be dissolved in a solution that is free of serum, because serum could contain esterases that would hydrolyze the compound.

A note on CF™ dye succinimidyl ester stability
Succinimidyl esters are generally susceptible to hydrolysis, which can result in lower labeling efficiency. Heavily sulfonated dyes, such as the Alexa Fluor® dyes, DyLight® dyes and IRDyes® are particularly hygroscopic, worsening the hydrolysis problem. For example, the percent of active Alexa Fluor® 488 succinimidyl ester (SE) could be well below 50% by the time of application (according to the manufacturer’s product datasheet). In a number of Alexa Fluor® SE reactive dyes, the SE group is derived from an aromatic carboxylic acid, while in all of Biotium’s CF™ dyes the SE group is prepared from an aliphatic carboxylic acid. This structural difference reduces the susceptibility of CF™ dye SE reactive groups to hydrolysis, resulting in relatively stable reactive dyes with consistently higher labeling efficiency compared to other SE derivatives of other fluorescent dyes.

Maleimides, MTS and thiosulfate dyes
Like the succinimidyl ester dyes, these dyes are also susceptible to hydrolysis, although generally to a much lower degree. Thus, for long term storage, anhydrous DMSO is recommended for making stock solutions.

Other reactive dyes
Amines, aminooxy (also known as oxylamine), hydrazide, azide, alkyne, BCN, and tyramide reactive dyes, as well as dye free acids, are generally stable in aqueous solution when stored at -20°C for 6-12 months or longer, as long as no compounds are present that may react with the dye’s functional group. See the product information sheets for specific reactive dyes more information.

Coelenterazines and D-luciferin

Coelenterazines are stable in solid form when stored as recommended; they are not stable in aqueous solution. Concentrated coelenterazine stock solutions (typically 1-100 mg/mL) should be prepared in ethanol or methanol; do not use DMSO or DMF to dissolve coelenterazines, because these solvents will oxidize the compounds. Ethanol or methanol stocks of coelenterazine can be stored at -20°C or below for six months or longer; alcohol stocks may evaporate during storage, so use tightly sealing screw cap vials and wrap the vials with Parafilm for long term storage. Propylene glycol also can be used as a solvent to minimize evaporation. If the solvent evaporates, the coelenterazine will still be present in the vial, so note the volume in the vial prior to storage so that you can adjust the solvent volume to correct for evaporation if needed. Prepare working solutions in aqueous buffers immediately before use. Coelenterazines are stable for up to five hours in aqueous solution.

Aquaphile™ coelenterazines are water soluble formulations of coelenterazines. They are stable in solid form when stored as recommended. Aquaphile™ coelenterazines should be dissolved in aqueous solution immediately before use. They are stable for up to five hours in aqueous solution.

Note that coelenterazines are predominantly yellow solids, but may contain dark red or brown flecks. This does not affect product stability or performance. If your coelenterazine is uniformly brown, then it is oxidized and needs to be replaced.

D-luciferin is stable in solid form and as a concentrated stock solution when stored as recommended; it is not stable at dilute working concentrations in aqueous solution. Prepare concentrated D-luciferin stock solutions (typically 1-100 mg/mL) in water, and store in aliquots at -20°C or below for six months or longer. Prepare working solutions immediately before use.

Most of our products are stable at room temperature for many days, but we recommend storage at 4°C or -20°C to prolong shelf life. In the case of many of our aqueous dye solutions, the compounds are very stable at room temperature, but we recommend cold storage to prevent the growth of mold or other microbes over time. Therefore, to save on shipping costs, products with recommended storage at 4°C or -20°C may ship at ambient temperature without affecting product performance. When you receive the product, place it under the recommended storage conditions.

Coelenterazines are predominantly yellow solids, but may contain dark red or brown flecks. This does not affect product stability or performance. If your coelenterazine is uniformly brown, then it is oxidized and needs to be replaced.

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