Services offered by the platform

The platform offers you different Thermal Shift Assay tests for your protein depending on the parameters you wish to study:

  • Feasibility test and optimization.
  • Screening of buffers for optimal thermostability.
  • Study of thermostability as a function of pH.
  • Influence of mutations and deletions on thermostability.
  • Measurement of protein•ligand interactions.
  • Screening and optimization of ligand interactions.
  • Kinetic stability measurement at different temperatures.
  • Study of protein•peptide or protein•protein interaction.
  • Stability measurement of protein•nucleic acid assembly and viral particles.

In order to check the compatibility of your sample with the tests offered in Thermal Shift Assay, the feasibility and optimization test must be carried out for all new proteins before the other services offered by the platform. Indeed, this first step will determine the feasibility of the project for the other desired services.

 

CTPF Plateform

Manager : Eric Jacquet (01 69 82 46 24)
Plateform engineer : Naima.Nhiri

Feasibility test and optimization

This test is necessary for any new protein studied by this technique on our platform. This test aims to verify that the protein sample is suitable for this application. It will allow us to determine the optimal concentrations of protein and fluorescent reagent. It is advisable to do these first thermostability tests in the protein storage buffer, so a few milliliters of this buffer should also be provided.

Buffer screening

The objective is to determine the most favorable conditions for better thermostability of a protein. The value of the protein denaturation temperature will be measured under different conditions of pH, buffer, salt, etc. We offer a microplate model of 48 pre-established conditions in duplicate. These conditions include 4 buffer conditions (TrisHCl pH 8.5; Phosphate pH 7.5; Hepes pH 7.0; MES pH 6.0) with varying concentrations of NaCl, KCl, MgCl2 and Glycerol. Special conditions can be prepared upon request. The quantity of protein necessary to achieve this offer will be determined during the feasibility test.

Thermostability depending on pH

The objective of this test is to determine more precisely the optimal pH value conferring better thermostability of the protein. For these experiments, we propose the use of different 3-components buffer systems allowing testing in each system a range from pH 4 to pH 10 out of 10 to 20 different pH values ​​in the range studied. The quantity of protein necessary to achieve this offer will be determined during the feasibility test.

Influence of mutations

Point modifications or deletions of residues can strongly affect the thermostability of these proteins. Experiments comparing denaturation profiles make it possible to quickly evaluate the impact of these mutations on the structure, the exposure of hydrophobic regions and thermal stability under different experimental conditions. The quantity of protein necessary to achieve this offer will be determined during the feasibility test.

Protein•ligand interactions: characterization and screening

TSA makes it possible to highlight protein•ligand interactions; these interactions modify the thermostability of the protein by forming a more stable complex. Variations of the order of 2 to 20°C in the denaturation temperature can be observed depending on the nature and affinity of this interaction. The shift in the denaturation curve can be measured as a function of increasing concentrations of substrates or inhibitors. This technique is also of prime importance for molecule screening projects for the search for new ligands.

Kinetics of denaturation

These experiments allow the measurement of the conservation of a native structure of the protein, at a given temperature over long periods of time. They therefore make it possible to select favorable buffer conditions to preserve this protein in its native form. The experiment includes 2 different phases: the incubation phase at constant temperature followed by denaturation kinetics which confirms the native or non-native state of the proteins after incubation for several hours. The incubation temperature and the duration of the experiment will be adapted according to the projects.

Study of macro-complexes

We are also developing the Thermal Shift Assay study of protein macro-complexes and the stability of viral particles on the platform. Several phage models have already been tested and validated, within the framework of collaboration with research teams, for measurements of stability of capsids or phages by TSA.