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High-Throughput Differential Scanning Fluorimetry of GFP-Tagged Proteins

  • Alanna E. Sorenson
  • Patrick M. SchaefferEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2089)

Abstract

Differential scanning fluorimetry is useful for a wide variety of applications including characterization of protein function, structure–activity relationships, drug screening, and optimization of buffer conditions for protein purification, enzyme activity, and crystallization. A limitation of classic differential scanning fluorimetry is its reliance on highly purified protein samples. This limitation is overcome through differential scanning fluorimetry of GFP-tagged proteins (DSF-GTP). DSF-GTP specifically measures the unfolding and aggregation of a target protein fused to GFP through its proximal perturbation effects on GFP fluorescence. As a result of this unique principle, DSF-GTP can specifically measure the thermal stability of a target protein in the presence of other proteins. Additionally, the GFP provides a unique in-assay quality control measure. Here, we describe the workflow, steps, and important considerations for executing a DSF-GTP experiment in a 96-well plate format.

Key words

Fluorimetry High-throughput screening Thermal shift assay Green fluorescent protein Ligand binding Enzyme inhibitors Selective protein unfolding Drug discovery 

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Copyright information

© Springer Science+Business Media, LLC 2020

Authors and Affiliations

  1. 1.Molecular and Cell Biology, College of Public Health, Medical and Veterinary SciencesJames Cook UniversityDouglasAustralia

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