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Running an experiment

The following is an example of a TSA experiment, from which the acquired data can be analyzed with “Thermott”. Given the experimental system, similar experiments can be carried out by measuring other signals besides fluorescence. As long as the signal corresponds to temperature-induced protein property changes, “Thermott” can be successfully used to interpret the data.

Use any available real time thermocycler or device capable of raising the temperature of the sample and recording fluorescence (or other technique) signal related to change of protein state.

Example protocol for affinity determination by TSA

  1. Decide how many experimental data points (different ligand concentrations) are needed. Usually about 8 points. Increasing number of experiment data points can reduce the influence of experimental data errors and significantly improve data interpretation. Experiments should include at least one negative control experiment (protein without ligand).

  2. Prepare protein stock solution (optimal concentration depends on the particular system, usually it is within 2-20 µM range).

  3. Prepare ligand stock solution in water or buffer (for example, 0.4 mM). Follow up with 2-fold serial dilutions of ligand stock. Dilutions may vary depending on system or number of data points.

  4. Mix the protein and ligand solutions (from steps 2-3) at 1:1 ratio and distribute protein into measuring chambers/tubes/capillaries. Depending on the equipment, mixing can be done directly in the test tubes.

  5. Insert samples into the thermocycler and start the experiment. The thermocycler program can vary considering the particular system. Typically, the experiment consists of raising temperature from room temperature to about 95°C at the speed of 1°C per min, and measuring the reporter signal at each step (usually every 30 s).

  6. After the experiment, analyze the obtained data using “Thermott” as described in other chapters of this guide.

For more detailed information about running a TSA experiment please refer to the protocols and practices of your local laboratory.

Protein stability assays

If you are running an assay to determine best protein storage conditions, consider using just a few or one concentration of the reagent in question. For comparing protein stability, usually ΔTm is enough.

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