Menu
biotium logo
Antibody Finder
Menu

GloMelt™ 2.0 Thermal Shift Protein Stability Kit

A high-throughput fluorescence-based assay for protein stability screening with higher tolerance for detergents and other protein additives compared to commonly used Thermal Shift dyes.

Reference dye
Size
Catalog #
price
Qty
200 assays
2,000 assays
Clear selection

Add to Cart
This product is available by special order only.

Please fill in the inquiry form and we will contact you shortly.

Product Description

The GloMelt™ 2.0  Thermal Shift Protein Stability Kit is a high-throughput and cost efficient method for screening ligand binding of molecules or identifying stability changes from protein modifications. The assay measures protein thermal stability, also known as a thermal shift assay, Protein Thermal Shift™, differential scanning fluorimetry, or Thermofluor assay.

Applications

  • A high-throughput method for sample screening via protein thermal stability
  • Screen molecules (e.g. drug candidates, antibodies) for ligand binding
  • Identify protein stability changes from point mutations or other modifications

Features

  • Higher tolerance for detergents and other additives compared to commonly used thermal shift dyes
  • Green fluorescence optimal for qPCR instruments and ROX normalization
  • Suitable for wide pH range, low reaction volumes, or low protein concentrations
  • Highly soluble and stable in aqueous buffers
  • Includes recombinant mouse IgG1 control protein — ideal for therapeutic antibody research.
  • Perfect for high-throughput assays with low volumes and protein concentrations.

Kit Components

  • GloMelt™ Dye, 200X
  • Recombinant Mouse IgG1 Control, 5 mg/mL
  • ROX Reference Dye, 40 uM
    (33028-T/33028-1 only)

Spectral Properties

  • GloMelt™ Dye Ex/Em 468/507 nm
  • ROX Reference Dye Ex/Em 575/600 nm

The GloMelt™ 2.0 Thermal Shift Protein Stability Kit features Recombinant Monoclonal Mouse IgG1 for performing control reactions. Compared to the polyclonal goat IgG control provided in our original GloMelt™ kit, this recombinant mouse antibody is more relevant for therapeutic antibody research and generates higher signal and a sharper melt peak between for robust assay validation.

A Simple and Rapid Method for Sample Screening

 

Figure 1. Environmentally sensitive fluorescent dyes can be used to monitor the temperature dependent unfolding of a protein. The protein’s melting temperature (Tm) is a reporter of the protein’s thermal stability.

GloMelt™ dye undergoes fluorescence enhancement upon binding to hydrophobic regions of denatured proteins, and therefore 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.

Small molecule inhibitors of bromodomain proteins have shown therapeutic effects in cancer models. Here, a GloMelt™ thermal shift assay was performed on 10 ug bromodomain BRD2 in the presence of bromodomain inhibitors JQ1 or RVX-208. Inhibitor binding stabilized the protein as indicated by the shift of the melting curves.

GloMelt™ Dye vs. Competitors

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.

Higher Tolerance for Detergents

IgG melt curve plots in the presence of detergent. A thermal shift assay was performed on 20 ug IgG in the presence of 5X SYPRO® Orange or 1X GloMelt™ Dye, using a QuantStudio™ 5 qPCR system. The presence of detergent inhibited the SYPRO® Orange assay, but had little affect on the GloMelt™ curve.

Higher Tolerance for Reducing Agents

IgG melt curve plots in the presence of DTT. A thermal shift assay was performed on 25 ug IgG in the presence of 1X PROTEOSTAT® TS dye or 1X GloMelt™ Dye, using a QuantStudio™ 5 qPCR system. The presence of DTT drastically reduced the sensitivity of the PROTEOSTAT® assay, but had little affect on the GloMelt™ Dye. As expected, DTT reduced IgG thermal stability.

To learn more about GloMelt™ and see how it compares to competitor products, visit our GloMelt™ Technology Page.

SYBR is a registered trademark of Thermo Fisher Scientific. Proteostat is a registered trademark of Enzo Life Sciences.

Product Attributes

Size
200 assays, 2,000 assays
Reference dye
No ROX, With ROX
Excitation/Emission
468/507 nm

Documents, Protocols, SDS and COA

FAQs

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 (SE) are generally susceptible to hydrolysis, which can result in lower labeling efficiency. Many commercially available fluorescent dyes used for life science research are heavily sulfonated dyes which makes them particularly hygroscopic, worsening the hydrolysis problem. In addition, for several commercially available SE reactive dyes, the SE group is derived from an aromatic carboxylic acid, while the SE group in all of Biotium’s CF® Dyes 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.

For dyes or reagents that are supplied lyophilized (as solids), it is hard to compare quantities based on appearance of the dye in the tube, because during the lyophilization process the dye can dry down in different ways, either spread out all over the tube, clumped together, or coating the sides or bottom of the tube. Centrifugation of the tube may not help in collecting the dye solid to the bottom of the tube as this generally works for solutions. However, lyophilized solids are packaged based on highly accurate absorbance measurement of the reagent solution prior to drying, so the vial will contain the correct amount of dye.

Biotium ships all antibodies (primary, secondary and conjugates) at room temperature. We guarantee their quality and performance under these conditions based upon our stability testing. Antibodies were subjected to accelerated stability testing by storing them at various temperatures (4°C, room temperature, or 37°C) for 1 week to mimic simulated shipping conditions and tested in immunostaining experiments. All antibodies showed the expected brightness and specificity, even after storage at sub-optimal temperatures for a week or longer. You can also download our Product Storage Statement here.

In line with our goal to be more environmentally friendly by reducing the use of excess packaging, and lowering shipping costs for our customers, products that have passed our stability testing are shipped at room temperature.

Once you have received the antibody vial, please follow the long-term storage instructions on the product information (PI) sheet.

Can’t find your answer?

Contact Us