I am using Roche Lightcycler 96 machine to perform protein thermal shift assays. I added 15 uL of 50 uM protein and 5 uL of SYPRO ORANGE fluorescent dye in DMSO. The temperature ranged from 30 °C to 90 °C，and the excitation/emission wavelengths are 470/514 nm or 533/572 nm. I could not see the melting of the protein, only a plain or descending curve. I wonder what is wrong with my thermal shift experiments. Thank you!
Dear Zhongtian Yu
just 2 questions:
1) 5ul of syproorenge 5000X original solution? or of a diluted stock?
2) Did you run also a positive control? eg a protein that you know is folded and it has to provide you a good transition?
In my experience the discending curve is typical of aggregate/unfolded proteins which are already unfolded and do not show the positive transition.
You can see an example of it at the minute 2' 30'' of the folliwing video aboit DSF
https://www.blogger.com/video.g?token=AD6v5dzr9KqZslTx9NeEjXx1XyPti8dU15eqYoE9TT8OEDaZQcNINuDKwzCAzVXkFWZA0btipKTr81doABh-VjpprwCcRukVcLhAXE_T5BzPxYbeQQA7VMFmc4DdpjS4XbOfxkwAhIWu
ProteoCool n°24 available on my blog ProteoCool (https://proteocool.blogspot.com/)
where i acquired the DSF profile for a protein unfolded by DTT addiction.
best regards
Manuele
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