Triosephosphate isomerase from Pyrococcusfuriosus forms numerous partially-unfolded structural states upon heating in the presence of denaturants. In this paper, we develop and deploy an electrophoretic technique based on SDS-PAGE chromatography performed with samples being loaded without boiling in the presence of SDS to demonstrate that different conformers can be trapped, visualized and quantitated densitometrically using SDS-PAGE electrophoresis to follow the progression of a protein unfolding reaction, and also to visualize partially-unfolded intermediate forms of proteins. The key novelty here is that hyperthermophile-derived proteins and their partially-unfolded forms are so kinetically stable that they fail to unfold in the presence of SDS in a gel sample-loading buffer if the sample is not boiled. This allows structural states to be trapped, separated and visualized on gels, because each has a different hydrodynamic volume which causes it to have a different mobility on the gel.

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