I am trying to calculate Tm of a kinase which is a dimer itself. I do scan from 4 to 90 deg at 1deg/min rate and around 50 deg I see a the line is going up but then it doesn't come down. i have tried using 5mM TCEP but nothing changes. i m using 2.5 mg/ml conc of protein in 50mM HEPES 50mM KCl 20% glycerol buffer. any suggestion? I have attached the scan image herewith. red one is my protein 1st scan and pink is the second scan.
First, what is the green line? Second, what kind of peak you want to see? Melting or something? A material in a solution is normally in a kind of melting state. Is kinase a liquid or a solid, without your buffer system?
Your first run looks like a glass transition temperature approximately 62°C. That is the step in your curve, assumed that your y-axis show endo up (it is important for DSC diagrams to say in which direction are endothermic or exothermic heat). That the line comes not back is totally normal after a Tg because the heat capacity increase during the transition region. Your second run looks like, that the Tg could be shifted to higher temperature, that could be an evidence for denaturation of your kinase during the heating if endo up.
Additionally, amorphous material has no Peaks, because this material is not melting you can not find a peak on amorphous material. In each temperature range the material is a liquid, but that is to complicated to explain this in this context.
I think that you have a peak around 62-63°C, but you should subtract a baseline, a scan of the buffer, with DSC origin for example. If you make this analysis you will see better the Tm of your protein.
Thank you everyone. I actually got a peak after baseline substraction. But I also did the same exp with 0.2 deg/min scan rate and the Tm came down to 43 deg ! Have any of you got this kind of experience before?
The position of the temperature depends on the heat rate. An increase in heat rate increase the position of the Tm peak. That depends on the inertia of the system and is the reason, why you have to say what your haet rate was to compare you results with other experiments.
Usually you can fins some differences with different scan rates, but 20°C is so much, are you sure you don't have any problem with your protein?
The question is, how the peak looks now for her first run with the baseline substraction and how the peak looks with the low rate. Because, in the uploaded picture, the peak is so broad and the peak high would be at 50°C not at 62-63°C.
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