GloMelt™ dye can be used to detect protein unfolding or measure thermal stability by performing a thermal shift assay, also called Protein Thermal Shift™, differential scanning fluorimetry, or Thermofluor assay.
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.
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 email@example.com for assistance.
Chemical products with special stability considerations:
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.
Some of our large volume light-sensitive products are packaged in amber bottles or amber glass vials, which are easy to handle and protect the products from light. However, amber micro packaging vials (0.5 mL or 2 mL) make it very difficult to see small quantities of dye when preparing solutions or pipetting. Therefore, we only package photoreactive dyes like PMA or PMAxx™ in amber micro packaging vials. Some of our reactive dyes are packaged in transparent vials, then sealed inside a moisture-resistant foil bag, which also will protect them from light.
Other fluorescent dyes in transparent vials should stored in the dark for long term storage. It’s fine to store dye vials uncovered in a windowless refrigerator or freezer with an automatic light shut-off. If dyes will be stored in a glass-front (deli-style) refrigerator, walk-in refrigerator/freezer, or at room temperature with constant light exposure, they should stored in a non-transparent box (like a white cardboard freezer box), in a closed drawer, in a black plastic bag, or covered with aluminum foil.
Fluorescent dyes generally are not sensitive to brief light exposure while they are being handled on the bench during an experiment. To be on the safe side, we usually loosely cover tube racks with a piece of foil if the dye vials are going to be out on the bench for more than 30 minutes or so. But most dyes are stable enough that even accidentally leaving them on the bench for a day will not affect performance. When handling photoreactive dyes like EMA, PMA, or PMAxx, we take the extra precaution of dimming the lights.
GloMelt™ dye undergoes fluorescence enhancement upon binding to hydrophobic regions of denatured proteins. Therefore the dye can be used to detect protein unfolding or measure thermal stability by performing a thermal shift assay, also called Protein Thermal Shift™, differential scanning fluorimetry, or Thermofluor assay.
The thermal shift assay is a rapid and inexpensive technique that quantifies change in protein denaturation temperature, and thus can be used to screen conditions that affect protein thermal stability, such as protein mutations, ligand binding, and buffer formulations (like pH, salts, detergents, and other additives). These assays are rapid (typically about 30 minutes) and are performed on a quantitative PCR system. The thermal shift method is compatible with high throughput screening and requires much less protein than methods such as circular dichroism and differential scanning calorimetry.
Optimize buffer formulation for protein stability and storage
Determine how mutations affect your protein’s stability
Rapidly screen small molecule drug candidates and other ligands for protein binding
GloMelt™ dye has significant advantages over other environmentally sensitive dyes, such as SYPRO® Orange and PROTEOSTAT® TS dye. GloMelt™ dye generates a strong signal because it is optimized for detection in the SYBR® Green channel of qPCR instruments, and therefore low reaction volumes and low protein concentrations can be used. GloMelt™ dye is compatible with high concentrations of protein stabilizers (such as glycerol and sorbitol), and also protein destabilizers (such as DTT and imidazole). GloMelt™ dye performs very well in high detergent concentrations, unlike SYPRO® Orange. Another advantage is that ROX dye can be included with GloMelt™ dye during thermal shift assays, which improves results by increasing replicate consistency in PCR instruments that require ROX passive reference dye.
GloMelt™ Thermal Shift Protein Stability Kit
GloMelt™ Dye, 200X
Goat IgG Control, 10 mg/mL
GloMelt™ Kit (with ROX):
GloMelt™ Dye, 200X
Goat IgG Control, 10 mg/mL
ROX Reference Dye, 40 uM
Protein Thermal Shift and QuantStudio are trademarks and SYBR is a registered trademark of Thermo Fisher Scientific. Proteostat is a registered trademark of Enzo Life Sciences.
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