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

[udubson]

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.

 

[ProfuselyQuarky]

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

 

[udubson]

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?

 

[ProfuselyQuarky]

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.