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General

You can follow this procedure as described in the following paper.  The analysis tool featured in the publication can be found from this link.

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.

Instrument Setup

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.

It is important that the data collection be turned on for the ramping portion of the melt curve. See an example run method below, noticing the status of the data collection icons.

Software Data Analysis

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.

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).

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.

We provide the 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 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.

The high background noise can stem from several different variables. A set of proper controls can narrow down the possible causes, which can be used to find the appropriate resolution. Below is a list of common causes of high background noise, and a resolution to the problem when the list of possible causes is narrowed:

Possible Causes

Symptom

Recommended Action

Native protein has external hydrophobic sites

High initial background signal and/or a small transitional increase in signal

  • The protein may not be a suitable candidate for Protein Thermal Shift™ studies.
  • Perform protein:dye titration studies to optimize the protein concentration and protein:dye ratio.

Protein solution contains high levels of detergent (>0.02%)

High initial background signal

High fluorescence in NPC wells

  • Perform protein:dye titration studies to optimize the protein concentration and protein:dye ratio.
  • Repurify the protein using an ammonium sulfate precipitation method. Resolubilize the purified protein using HEPES buffer or a buffer with neutral pH, then add glycerol and DTT.

Buffer component interacts with the dye

High initial background signal

High fluorescence in NPC wells

High fluorescence in LOC wells

  • Perform protein:dye titration studies to optimize the protein concentration and protein:dye ratio.
  • Perform a buffer screening Protein Thermal Shift™ study to identify alternative buffer conditions.

Ligand interacts with the dye

High initial background signal

High fluorescence in LOC wells

Use an alternate method to screen for conditions that affect thermal stability of the protein.

Protein aggregation or the protein is partially unfolded

High initial background signal

Flat signal or decrease in signal

  • Repeat the study with a fresh protein sample.
  •  Perform a buffer screening Protein Thermal Shift™ study to identify buffer conditions that increase thermal stability of the protein, then repeat the original study using the new buffer conditions.

Make sure that you first opened the file in the corresponding instrument software first, clicked “Analyze”, and then saved 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.

Some proteins have some hydrophobic residues on the surface and the dye binds to this. When we heat it up, the protein unfolds and more hydrophobic residues are 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. Contact us about the possibility of other dyes being available for this issue.

We recommend looking at the spread in Tm, which is more important than the relative fluorescence.