Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

Robert P. Sparks, William Lawless, Andres S. Arango, Emad Tajkhorshid, Rutilio A. Fratti

Research output: Contribution to journalArticlepeer-review

Abstract

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane trafficking, signal transduction and cytoskeletal remodeling. Classic tools for measuring such interactions include surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). While powerful tools, these approaches have setbacks. ITC requires large amounts of purified protein as well as lipids, which can be costly and difficult to produce. Furthermore, ITC as well as SPR are very time consuming, which could add significantly to the cost of performing these experiments. One way to bypass these restrictions is to use the relatively new technique of microscale thermophoresis (MST). MST is fast and cost effective using small amounts of sample to obtain a saturation curve for a given binding event. There currently are two types of MST systems available. One type of MST requires labeling with a fluorophore in the blue or red spectrum. The second system relies on the intrinsic fluorescence of aromatic amino acids in the UV range. Both systems detect the movement of molecules in response to localized induction of heat from an infrared laser. Each approach has its advantages and disadvantages. Label-free MST can use untagged native proteins; however, many analytes, including pharmaceuticals, fluoresce in the UV range, which can interfere with determination of accurate KD values. In comparison, labeled MST allows for a greater diversity of measurable pairwise interactions utilizing fluorescently labeled probes attached to ligands with measurable absorbances in the visible range as opposed to UV, limiting the potential for interfering signals from analytes.

Original languageEnglish (US)
Article numbere60607
JournalJournal of Visualized Experiments
Volume2022
Issue number180
DOIs
StatePublished - Feb 2022

ASJC Scopus subject areas

  • General Chemical Engineering
  • General Immunology and Microbiology
  • General Biochemistry, Genetics and Molecular Biology
  • General Neuroscience

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