Dear Samer

in my experience high initial fluorescence means that the protein is partially unfolded and/or aggregate and it can already bind sypro orange at low itemperature.

For example i obtained a similar profiles when i compared the dsf of a folded protein which contain 2 S-S in prensece and absence of dtt.

As expected, addiction of the reducing agent, destroid the s-s and we obtained an increase in initial fluorescence simiar to your, while with out dtt the dsf profile was perfect whith a strong transition around 53°C

i think that in your case is possible that:

- you have a mix unfolded and folded protein and the unfolded cover the profile of the folded.

- you protein contain 2 domains, 1 folded that bine sypro orange from the beginning and 1 more folded that is responsabile for the sliglty change in fluorescence that you are around 45°C.

which is True?

try to perform a sec to see if you have aggregates or if you can move to dsc.

good luck

Manuele

Thank you Manuele. That makes sense. Do you think the curves provided are of sufficient quality to continue using this same protein stock to see if different ligands cause a shift in the melting curve (Since I still get melt peaks when the dF/dT vs temp. graph is made by the software)?

Thanks,

Samer

Manuele Martinelli