Hello All,
I am doing a protein-peptide thermal shift assay in QuantStudio3. I have observed that most of the peptides are giving a significant negative shift in Tm, though some are giving a positive shift. As per the literature, the ligands/peptide giving more than 2-degree positive shift in Tm are considered as hits. But what is the significance of negative shift here?
Other questions are why few peptides are giving high fluorescence signal significantly enough to overshadow the reference? I am using the manufacture’s PTS dye. Would another dye help in this case?
Thank you very much.
Hi
There are several papers discussing negative shifts as binders at least for fragment-protein binding as much I can remember. From Chris Abel group.
Otherwise it could be aggregation or interference. How does your baseline look like? If your peptides are giving strong fluorescence then this could be a factor.
I cant agree with the idea that deltaTm more than 2 degrees is a hit. There is no direct correlation between DeltaTm and KD. I think you need to define what is " a hit" and what are you trying to look at using DSF.
If you screen a whole library and are trying to enrich your library for other orthogonal methods then DSF can help but if you have a handful of peptides then maybe you should be looking at using other methods.
Hi Mostafa,
Thanks for the response. Yes, I am trying to enrich the library for orthogonal assay. It is true that DSF can not give KD but can enrich the library for possible positive hits.
ok then seems you're on the right path.
For negative ones I wouldnt discard them as there are a few papers on PPI inhibitors which discuss their importance
Mostafa Jamshidiha Hi, can you give references to those papers. I also had some negative shifts for a library of compounds and I am trying to understand the reasoning behind. Thanks a lot in advance!
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