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View additional product information for Protein Thermal Shift™ Starter Kit - FAQs (4462263)
27 product FAQs found
We recommend looking at the spread in Tm, which is more important than the relative fluorescence.
Some proteins have hydrophobic residues on the surface and the dye binds to these residues. Heating results in unfolding of the protein causing more hydrophobic residues to be exposed. The dyes bind preferentially to these inner locations and so there is a flattening (or a very low rise) of the melt discernable in the melt profile. If there is no positive slope, you will not get a Boltzmann Tm, but you should still get a derivative one. And you can always draw a manual region to get a Tm out. Some proteins will not work with this technology if the hydrophobic residues are already exposed on the surface and the dye binds strongly to it. Please contact Technical Support at techsupport@thermofisher.com about the possibility of other dyes being available for this issue.
Make sure that you first open the file in the corresponding instrument software, click “Analyze”, and then save the file, before trying to open with the Protein Thermal Shift Software. The file must first be analyzed before it can be used in the Protein Thermal Shift Software.
The software will allow for ≥100 plates per study. We allow the user this flexibility but do not recommend you mix data from multiple plates unless they have validated their results in advance. At a minimum, we recommend researchers include a reference assay in each plate to ensure reproducibility.
We provide two independent methods because they each have unique things to offer in terms of the analysis. The two-state Boltzmann model has a physical meaning and appeal. It also provides a great way to normalize across noisy undulations in the signal. However, those undulations may be of actual interest and not noise, such as for multi-domain proteins where they may correspond to different domains coming apart in stages. Here the two-state model is inappropriate. The derivative method can help get a temperature at which the local peaks occur. These are two completely unrelated approaches. If the two-state model is a great fit for your data, the results should be in close agreement.
The Boltzmann is a two-parameter model for the transition between the two states (i.e., the native and unfolded configurations of the protein). The Boltzmann equation is a two-state sigmoidal curve. The start and end region should be chosen such that the interim signal best resembles a sigmoidal profile. So the start should be where the signal is still relatively flat, and the end should be where the signal has already risen to its highest level.
The Protein Thermal Shift Software will only accept and analyze data files (*.eds) generated from compatible Applied Biosystems Real-Time PCR instruments (QuantStudio, ViiA 7, 7500/7500F (with SDS v2.0.x), or the StepOne/StepOnePlus systems).
We recommend 3-4 technical replicates, and the Tm spread of replicates will depend on how sensitive the protein is to manipulation. A good set of replicates will have a range of <0.5°C, with <0.1°C for most well-behaved proteins.
It is important that the data collection be turned on for the ramping portion of the melt curve.
Choose “Run Method” under the “Setup” tab in the left hand side of the ViiA 7 Software. Choose the tab, “Optical Filters” and an option to choose the excitation and emission filter will be shown. Select the X1-M3 filter combination.
Buffers do impact the binding of a ligand, and it is usually best to choose a buffer that increases the Tm. In addition, you must consider which buffer is more representative of the environment where the protein and ligand would exist (for example, blood, plasma, etc.). We recommend trying a few buffer conditions, as the Protein Thermal Shift technology is conducive to screening many conditions within a short amount of time.
You can follow the procedure described in this paper (http://www.ncbi.nlm.nih.gov/pubmed/17853878). The analysis tool featured in the publication can be found from this link (ftp://ftp.sgc.ox.ac.uk/pub/biophysics/).
The Protein Thermal Shift Buffer is a neutral (pH 6.8) potassium phosphate-based buffer.
The Protein Thermal Shift Control Protein is supplied at 1 µg/µL. Guidance on how to set up the reaction components for the protein melt reaction can be found on page 36 of the user guide: https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0025600_Protein_Thermal_Shift_UG.pdf
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.
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.
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.
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 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
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.
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.
We provide the two independent methods because they each have unique analysis offerings. The two-state Boltzmann model has a physical meaning and appeal. It also provides a way to normalize across undulations in the signal. However, those undulations may be of actual interest and not noise. For example, for multi-domain proteins they may correspond to different domains coming apart in stages. For this, the two-state method is inappropriate. The derivative method can help determine the temperature at which the local peaks occur.
The optimal concentration should be determined empirically, but the average input is ~1 µg per well, with a range of 0.05–5 µg of protein.
Please consult the Release Notes for the Protein Thermal Shift Software for the latest compatibility information. Below are the respective instrument and software compatibility for use with the Protein Thermal Shift Software v1.4
- ViiA 7 Real-Time PCR System with ViiA 7 Software v1.0 and later
- QuantStudio 6 and 7 Flex with QuantStudio Real-Time PCR Software v1.0 or later
- 7500/7500F Real-Time PCR System with v2.0.4 and later
- StepOne and StepOnePlus Real-Time PCR Systems with StepOne Software v2.1 and later
- QuantStudio 12K Flex Real-Time PCR System with QuantStudio 12K Flex Software v1.0 and later
- QuantStudio 1 Real-Time PCR System with QuantStudio Design and Analysis Software v1.5 and later
- QuantStudio 3 and 5 Real-Time PCR System with QuantStudio Design and Analysis Software v1.2 and later
- QuantStudio 6 Pro or 7 Pro Real-Time PCR System with QuantStudio Design and Analysis Software v2.0 and later (desktop version and Connect cloud-based version)
The chemistry behind Protein Thermal Shift Dye is proprietary.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
The Protein Thermal Shift Control Ligand is provided at a concentration of 100 mM. Guidance on how to set up the reaction components for the protein melt reaction can be found on page 36 of the user guide: https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0025600_Protein_Thermal_Shift_UG.pdf
The Tm of the control protein will vary by instrument, how well the protein has been treated, concentration, buffer used, etc. The Tm range for the control protein is about 41-47 degrees C, dependent on conditions. However, your experiment should have its own “control” or reference protein/conditions, which you will need to set up.