| Autor: |
Sorenson AE; Molecular and Cell Biology, College of Public Health, Medical and Veterinary Sciences, James Cook University, Douglas, QLD, Australia., Schaeffer PM; Molecular and Cell Biology, College of Public Health, Medical and Veterinary Sciences, James Cook University, Douglas, QLD, Australia. patrick.schaeffer@jcu.edu.au. |
| Jazyk: |
angličtina |
| Zdroj: |
Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2020; Vol. 2089, pp. 69-85. |
| DOI: |
10.1007/978-1-0716-0163-1_5 |
| Abstrakt: |
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. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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