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Experimental Setup

For initial experiments, we suggest the following ranges as a starting point:
Protein Thermal Shift™ Dye: Use the dye at 1X–20X
Total volume per reaction: 10–50 µL
Total protein per reaction: ~0.05–5 µg (average 1 µg)
Typical thermal profile: 25°C–99°C

Set up the reaction on ice and start the run as soon as convenient. The Protein Thermal Shift™ Dye stability at 4°C and room temperature is at least 24 hours, in the dark.

No; the assay works on most proteins we have tested (by some estimates up to 90%), but it may fail if detergents (sometimes used to solubilize membranes) are present in the buffer, if the protein does not have enough hydrophobic residues to provide a strong fluorescent signal (i.e., small proteins), or if the protein has too many exposed hydrophobic residues, resulting in high background fluorescence (might be the case for large macromolecular complexes). Each protein will require different optimal conditions; however, most proteins are successful with the first set of screens.

The Protein Thermal Shift™ Dye can be diluted in either water or buffer. If you are performing a buffer screening experiment, use water or a neutral buffer. For small-molecule/ligand screening, use the same buffer that is being used in the screen. For protein stability analysis, dilute in the buffer the protein is stored in. It is also important to make a fresh 8X stock each time.

Our instruments can go down to 4°C for the melt stage. Please note the holding temperature is 25°C, so if you want to start a melt at a lower temperature, you need to keep your plate on ice, put it into the instrument, and start the run right away with the maximum ramp rate to bring the temperature down.

The amount of protein varies depending on the type of protein and the experimental conditions. We recommend screening using approximately 0.05–5 μg (1 μg on average) in a total volume of 10–50 μL to determine the optimal amount of protein needed for your experiments.

Reagents

No additional calibrations are required (it uses the ROX™ dye calibration profile) to use the Protein Thermal Shift™ Dye, as it works using standard melt protocols on Applied Biosystems® instruments.

Yes. However, the Protein Thermal Shift™ dye for membrane proteins is not available as an inventoried item and is available only as a custom product. Please contact your local sales representative for more information.

The Dye Kit only provides Protein Thermal Shift™ Dye and Protein Thermal Shift™ Buffer, whereas the Starter Kit includes the Protein Thermal Shift™ Dye and Protein Thermal Shift™ Buffer, as well as a Protein Thermal Shift™ Control Protein and Protein Thermal Shift™ Control Ligand, which can be used to get acquainted with the application. We recommend that you use the Starter Kit first to learn the basics of the assay, and order the Dye Kit when starting with your own experiments.

The Protein Thermal Shift™ Control Ligand is supplied at 10 mM, and the Protein Thermal Shift™ Control Protein is supplied at 1 µg/µL.

The Protein Thermal Shift™ Buffer is a neutral, potassium phosphate–based buffer.

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