I am conducting a study on DnaK with four compounds that are known inhibitors of human Hsp70. As part of this, I performed thermal denaturation assays at 20°C, 40°C, 60°C, 80°C, and 100°C to observe DnaK's behavior in the presence and absence of these compounds. The experimental phase is complete, and I have obtained all spectroscopic data, including the highest peak wavelength for each temperature. However, I am having difficulty normalizing the data on a scale from 0 to 1, where 1 represents 100%. Initially, I planned to use the highest peaks at 20°C and 100°C as my baseline for 1 and 0, but this approach hasn't worked as expected. I need assistance in understanding how to properly normalize this data
Gudani Tshiswaise
The simplest way is to plot out intensity of each wavelength as a function of temperature. See how does it look. If the unfolding reaction follows two-state scenario, you will see a sigmoidal transition at certain wavelengths.
A sophisticated way is singular value decomposition (SVD) analysis, which will take all wavelengths into account and result in the most representative components that can be used for normalization and derived the folded fraction.
See Figure S4 in the following reference:
https://www.frontiersin.org/journals/chemistry/articles/10.3389/fchem.2021.663241/full#supplementary-material
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