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Anal Biochem. 2016 Sep 1;508:114-7. doi: 10.1016/j.ab.2016.05.019. Epub 2016 Jun 11.

Native denaturation differential scanning fluorimetry: Determining the effect of urea using a quantitative real-time thermocycler.

Author information

1
Carleton University, Department of Biology, 1125 Colonel By Drive, Ottawa, Ontario K1S 5B6, Canada.
2
Carleton University, Department of Biology, 1125 Colonel By Drive, Ottawa, Ontario K1S 5B6, Canada. Electronic address: kenneth_storey@carleton.ca.

Abstract

The effect of protein stability on kinetic function is monitored with many techniques that often require large amounts of expensive substrates and specialized equipment not universally available. We present differential scanning fluorimetry (DSF), a simple high-throughput assay performed in real-time thermocyclers, as a technique for analysis of protein unfolding. Furthermore, we demonstrate a correlation between the half-maximal rate of protein unfolding (Knd), and protein unfolding by urea (I50). This demonstrates that DSF methods can determine the structural stability of an enzyme's active site and can compare the relative structural stability of homologous enzymes with a high degree of sequence similarity.

KEYWORDS:

Differential scanning fluorimetry; Enzyme kinetics; Enzyme stability; Lactate dehydrogenase; Native denaturation; Urea

PMID:
27296634
DOI:
10.1016/j.ab.2016.05.019
[Indexed for MEDLINE]

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