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.
The storage conditions that a Biotium label recommends are deliberately conservative. Long term storage at low temperatures usually will increase the time that the product will remain within specification, unless specific instructions otherwise are provided (such as “do not refrigerate” or “do not freeze”).
Biotium labels and product information sheets may show a specific temperature for long term storage, or a temperature range that is adequate for long term storage. For all products, the implied range is as follows for the following storage conditions:
For Biotium products where the label indicates room temperature or RT, this implies storage in ambient conditions between 20°C and 30°C.
For Biotium products where the label indicates 4°C, this implies storage in a refrigerator that is between 2°C and 8°C.
For Biotium products where the label indicates -20°C, this implies storage in a freezer that is between -35°C and -5°C.
Ultra Low Temperature Freezer
For Biotium products where the label indicates -70°C, this implies storage in an ultra low-temperature (ULT) freezer that is below -60°C.
Compound solubility is listed on product web pages, product information sheets or product safety and data sheets. Check the product information sheet for specific instructions for preparing stock solutions. Stock solutions are generally prepared at 10X-1000X the final required working concentration, typically in the range of 1-100 mg/mL. For dyes and indicators, we generally prepare a concentrated stock solution at 5-10 mM in the recommended solvent. The dye can then be diluted to the final desired concentration in the buffer or medium used for the application.
> 1000 mg/mL
Very Slightly Soluble
< 0.1 mg/mL
To dissolve lyophilized compounds, simply add the appropriate volume of the recommended solvent to the vial to make the desired concentration stock solution, and swirl or gently vortex to mix. Make sure the solvent comes in contact with the inside walls of the vial to fully recover the product.
Difficult to dissolve compounds can be heated to 50°C or higher (note: we do not recommend heating coelenterazine solutions), and vortexed or sonicated until completely dissolved. Some chemical compounds are just kinetically slow to dissolve. This can be especially true when the compounds are highly pure and in crystalline form. An alternative and gentler way to dissolve these compounds can be to simply rock the compound/solvent mixture in the dark overnight.
When preparing stock solutions in organic solvents such as DMF, DMSO, or alcohol for use in living cells or organisms, make a concentrated stock so that the final concentration of solvent in the working solution will not be toxic. A general guideline for immortalized cell lines is to keep the final solvent concentration below 1%; certain cell lines, primary cells or experimental organisms may be more sensitive to solvent toxicity. Similarly, for enzymatic reactions, the final concentration of solvent should be kept below 1% to avoid inhibiting enzyme activity.
Compounds dissolved in aqueous solution can be sterilized by filtration if necessary. Generally, concentrated stock solutions of compounds in organic solvents do not require sterilization, but culture medium can be sterilized by filtration after addition of the compound.
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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