Protocol for performing and optimizing differential scanning fluorimetry experiments

Taiasean Wu; Michael Hornsby; Lawrence Zhu; Joshua C Yu; Kevan M Shokat; Jason E Gestwicki

doi:10.1016/j.xpro.2023.102688

Protocol for performing and optimizing differential scanning fluorimetry experiments

STAR Protoc. 2023 Dec 15;4(4):102688. doi: 10.1016/j.xpro.2023.102688. Epub 2023 Nov 8.

Authors

Taiasean Wu¹, Michael Hornsby², Lawrence Zhu³, Joshua C Yu¹, Kevan M Shokat⁴, Jason E Gestwicki⁵

Affiliations

¹ Department of Pharmaceutical Chemistry and the Institute for Neurodegenerative Disease, University of California, San Francisco, San Francisco, CA 94158, USA.
² Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 941583, USA.
³ Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA.
⁴ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 941583, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA.
⁵ Department of Pharmaceutical Chemistry and the Institute for Neurodegenerative Disease, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address: jason.gestwicki@ucsf.edu.

Abstract

Differential scanning fluorimetry (DSF) is a widely used technique for determining the apparent melting temperature (Tma) of a purified protein. Here, we present a protocol for performing and optimizing DSF experiments. We describe steps for designing and performing the experiment, analyzing data, and optimization. We provide benchmarks for typical Tmas and ΔTmas, standard assay conditions, and upper and lower limits of commonly altered experimental variables. We also detail common pitfalls of DSF and ways to avoid, identify, and overcome them.

Keywords: Biophysics; High-Throughput Screening; Molecular/Chemical Probes; Protein Biochemistry.

MeSH terms

Amines*
Calorimetry, Differential Scanning
Fluorometry / methods
Proteins*
Temperature

Substances

Proteins
Amines

Grants and funding

R01 GM141299/GM/NIGMS NIH HHS/United States