CF® Dye Amine

The exact chemical structures of CF® dyes are currently confidential but will be fully disclosed at a later stage when pending patents become granted. In general terms, the structure of a CF® dye may be divided into two parts: a) dye core structure (i.e. the aromatic ring skeleton that defines the dye’s color or absorption/emission wavelengths), and b) core structure-modifying elements. At present, CF® dyes bear the core structures of coumarin, pyrene, rhodamine or cyanine dyes. Blue fluorescent CF® dyes are based on coumarin or pyrene dye core structure, while green to near-IR CF® dyes are based on either cyanine or rhodamine dye core structures. Core structure-modifying elements refer to various chemical attachments to the core structure and are a key aspect of the CF® dye invention that makes CF® dyes superior to other commercial dyes.

Category: CF® Dyes

Rhodamine-based CF® dyes (designated R) generally have better photostability but weaker fluorescence than their cyanine-based equivalents (designated C). Therefore, rhodamine-based near-IR CF® dyes are a better choice for microscopy, while cyanine-based CF® dyes are more ideal for flow cytometry, Western blotting, and other applications where photobleaching is less of a concern. Another factor to consider is the size of the dyes. Some of the cyanine-based near-IR CF® dyes are much larger than the rhodamine-based equivalents. For antibody labeling, either version of the CF® dyes is suitable. However, for applications where the dye size may cause a steric problem, the smaller dye may be a better choice.

Category: CF® Dyes

All three of these dyes can be excited by the 405 nm laser (or UV mercury lamp). They differ in their emission wavelengths. CF®405S has blue fluorescence emission at 431 nm, similar to AlexaFluor® 405, Cascade Blue®, and DyLight™ 405 (Thermo Scientific). CF®405M has longer wavelength blue fluorescence emission at 452 nm, similar to Horizon™ V450 (BD Biosciences) and Pacific Blue® (Thermo Scientific). CF®405L has orange fluorescence emission at 545 nm, similar to Pacific Orange® (Thermo Scientific). We recommend choosing the dye that best fits your instrument’s detection settings. See our CF® Dye Quick Reference Table and CF® Dye Selection Guide for more information on CF® dyes.

Category: CF® Dyes

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.

Category: Luciferase Assays, Product shipping, storage, shelf life, & solubility

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.

Category: GelRed® and GelGreen® Nucleic Acid Gel Stains, CF® Dyes, Mix-n-Stain™ Antibody Labeling Kits, Apoptosis, Necrosis, and Cell Viability Kits, NucView® Caspase-3 Enzyme Substrates, DNA Quantitation Kits, Luciferase Assays, Product shipping, storage, shelf life, & solubility

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.

Category: CF® Dyes, Mix-n-Stain™ Antibody Labeling Kits, NucView® Caspase-3 Enzyme Substrates, DNA Quantitation Kits, Luciferase Assays, Product shipping, storage, shelf life, & solubility