Microscale Thermophoresis to Study Protein-Lipid Interactions in Solution

Samenvatting

Source: Sparks, R. P. et al., Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions. J. Vis. Exp. (2022)

This video demonstrates microscale thermophoresis for studying the protein-lipid interaction. The assay detects the interaction between molecules by quantifying the thermophoretic movement of fluorescent-labeled proteins in response to a temperature gradient. The fluorescent molecule is mixed with different concentrations of the non-fluorescent lipid molecules, and the mixture of molecules in the solution is loaded into capillaries. A temperature gradient is applied to the samples in the capillaries, and the movement of the fluorescent molecule in the temperature gradient is detected and recorded.

Protocol

1. Preparation of materials

1. Prepare phosphate-buffered saline (PBS): 137 mM NaCl, 2.5 mM KCl, 10 mM NaH₂PO₄, and 2 mM KH₂PO_4, pH 7.4.
2. Prepare NTA-Atto 647 N dye. Dilute stock NTA-Atto 647 N dye to 100 nM from a 100% DMSO solution into PBS without Tween.
3. Express Vam7-His8 – Protein as a fusion protein in E. coli and purify it using Ni-NTA and size exclusion chromatography.
4. Titrate the analyte in PBS buffer. If the analyte is in a different buffer, labeled MST is not particularly sensitive to minor buffer differences from molecules such as....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Cy5 Maleimide Mono-Reactive Dye	GE Healthcare	PA23031	For protein labeling
GraphPad Prism	GraphPad software
Monolith NT.115 Capillaries (1000 count)	Nanotemper	MO-K022	Capillaries for MST
Monolith NT.115 machine	Nanotemper		University equipment
NTA-Atto 647 N	Sigma	2175	Label for His tags
Phosphatidylinositol 3-phosphate diC8 (PI(3)P diC8)	Echelon	P-3008	Lipid for binding experiments