How to interpret thermal shift experiment's work for binding?

  • #1
udubson
2
1
Hello, PF I’m new here. Can someone please help me explain how to interpret thermal shift experiments work for binding? Apparently the data you receive from such experiment is a derivative dF/dT where F=fluorescence and T=temperature; and I’m very confused because isn’t the experiment supposed to help you determine when a protein begins to melt and denature? What does fluorescence have to do with this and does this mean you can only use proteins with GFP? I’m studying physics and proteomics is not my forte so apologies.
 
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  • #2
Do you understand qPCR? The assays’ method involves a dye that fluoresces during denaturation. The choice of dye is dependent on specific experiment and protein
 
  • #3
ProfuselyQuarky said:
Do you understand qPCR? The assays’ method involves a dye that fluoresces during denaturation. The choice of dye is dependent on specific experiment and protein
I would have to read more about it but I know it’s “real time PCR”. where does the derivative come to play exactly? And how does an inanimate dye fluoresce during denaturation?
 
  • #4
the dye interacts with protein residues that become exposed only when protein begins to unfold and only then does it fluoresce. So, more fluorescence corresponds with more unfolding. dF/dT is just the change in fluorescence with respect to temperature, so then you get very specific readings of what temp your protein begins to fold at.
 
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Related to How to interpret thermal shift experiment's work for binding?

1. What is a thermal shift assay and how does it work?

A thermal shift assay, also known as a differential scanning fluorimetry (DSF) or thermal stability assay, measures the stability of a protein as it is heated. The assay monitors the unfolding of the protein by detecting changes in fluorescence, typically using a dye that binds to hydrophobic regions exposed during unfolding. The temperature at which the protein unfolds, known as the melting temperature (Tm), can indicate protein stability and binding interactions.

2. How can I determine if a ligand is binding to my protein using a thermal shift assay?

When a ligand binds to a protein, it often stabilizes the protein structure, resulting in an increase in the melting temperature (Tm). By comparing the Tm of the protein alone to the Tm of the protein in the presence of the ligand, you can determine if binding has occurred. A significant shift in the Tm suggests that the ligand is interacting with the protein.

3. What controls should I include in my thermal shift assay?

Essential controls for a thermal shift assay include the protein alone (to establish the baseline Tm), the protein with a known ligand (to confirm the assay's sensitivity), and the ligand alone (to ensure it does not interfere with the fluorescence signal). Additionally, including a buffer-only control can help identify any background fluorescence or artifacts.

4. How do I interpret a non-significant shift in the melting temperature (Tm)?

A non-significant shift in the Tm could indicate that the ligand does not bind to the protein, or it binds with very low affinity. It is also possible that the binding does not significantly alter the protein's thermal stability. Other factors, such as experimental conditions or protein quality, could also affect the results. Further experiments, such as isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR), might be needed to confirm binding.

5. What are the limitations of using thermal shift assays for studying protein-ligand interactions?

Thermal shift assays are limited by their reliance on changes in protein stability upon ligand binding. Not all binding events result in detectable shifts in Tm, especially if the binding does not significantly stabilize the protein. Additionally, the assay requires a fluorescent dye that may not be compatible with all proteins or ligands. The method also provides limited information about the binding kinetics and affinity, necessitating complementary techniques for a comprehensive analysis.

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