Hi!
I am profiling a novel protein and one of the parameter is the effect of thermal exposure to the protein. For that, I plan to look into the effect on its activity which I already did. Next is to look at the physical changes upon thermal exposure.
Originally I plan to conduct circular dichroism analysis to assess on the secondary structure changes. Due to some constraint, I cannot do so. So Im looking for another method to look at the changes. Preferably some cheap and fast option.
I plan to use native PAGE to look at the changes from the band condition (smearing, sharp, size shifting) but is there any better option to really replace CD?
Differential scanning fluorescence and differential scanning calorimetry are standard methods that allow you to measure the thermally-induced denaturation of proteins.
If the protein has tryptophan residues, you could look at changes to the fluorescence emission spectrum and/or intensity, which are sensitive to the hydropathy of the environment surrounding the side chain. For example, a red shift of the UV emission peak indicates a more hydrophilic environment, which would be consistent with the initial stage of heat denaturation. Circular dichroism can also be used to study the effect of temperature on the environment of tryptophan residues.
Dynamic light scattering looks at the hydrodynamic radius of the protein molecules, which could change due to denaturation.
You can also do, size exclusion chromatography and limited proteolysis to check the quality of your protein after thermal treatment. Generally, high thermal exposure leads to the protein denaturation which causes protein hydrophobic core to get exposed. SEC will give some idea about the protein characteristics such as monomer, dimer or oligomer. Limited proteolysis with a specific protease will again tell you the protein regions that are accessible to protease in native v/s thermal exposed conditions.
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