Expression, purification, and characterization of anuran saxiphilins using thermofluor, fluorescence polarization, and isothermal titration calorimetry

Zhou Chen; Sandra Zakrzewska; Holly S. Hajare; J. Du Bois; Daniel L. Minor, Jr.

STAR Protocols (Mar 2024)

Expression, purification, and characterization of anuran saxiphilins using thermofluor, fluorescence polarization, and isothermal titration calorimetry

Zhou Chen,
Sandra Zakrzewska,
Holly S. Hajare,
J. Du Bois,
Daniel L. Minor, Jr.

Affiliations

Zhou Chen: Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94158-2330, USA
Sandra Zakrzewska: Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94158-2330, USA
Holly S. Hajare: Department of Chemistry, Stanford University, Stanford, CA 94305, USA
J. Du Bois: Department of Chemistry, Stanford University, Stanford, CA 94305, USA; Corresponding author
Daniel L. Minor, Jr.: Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94158-2330, USA; Departments of Biochemistry and Biophysics, and Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158-2330, USA; California Institute for Quantitative Biomedical Research, University of California, San Francisco, San Francisco, CA 94158-2330, USA; Kavli Institute for Fundamental Neuroscience, University of California, San Francisco, San Francisco, CA 94158-2330, USA; Molecular Biophysics and Integrated Bio-imaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Corresponding author

Journal volume & issue: Vol. 5, no. 1
p. 102792

Abstract

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Summary: Anuran saxiphilins (Sxphs) are “toxin sponge” proteins thought to prevent the lethal effects of small-molecule neurotoxins through sequestration. Here, we present a protocol for the expression, purification, and characterization of Sxphs. We describe steps for using thermofluor, fluorescence polarization, and isothermal titration calorimetry assays that probe Sxph:saxitoxin interactions using a range of sample quantities. These assays are generalizable and can be used for other paralytic shellfish poisoning toxin-binding proteins.For complete details on the use and execution of this protocol, please refer to Chen et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Published in STAR Protocols

ISSN: 2666-1667 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science: Science (General)
Website: https://star-protocols.cell.com/

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