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 Prepare phosphate-buffered saline (PBS): 137 mM NaCl, 2.5 mM KCl, 10 mM NaH2PO4, and 2 mM KH2PO4, pH 7.4. 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. Express Vam7-His8 – Protein as a fusion protein in E. coli and purify it using Ni-NTA and size exclusion chromatography. Titrate the analyt…