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Measurement of Protein-Protein Interactions through Microscale Thermophoresis (MST)

Abstract : The binding interactions of PD-1 and PD-L1 have been studied by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) over the past few years, but these investigations resulted in controversy regarding the values of the dissociation constant (Kd) (Freeman et al., 2000). MST is a powerful new method for the quantitative analysis of protein-protein interactions (PPIs) with low sample consumption. The technique is based on the movement of molecules along microscopic temperature gradients, and it detects changes in their hydration shell, charge or size. One binding partner is fluorescently labeled, while the other binding partner remains label-free. We used a protocol that allows the determination of the binding affinity by MST without purification of the target protein from the cell lysate. The application of this MST method to PD-1-eGFP and PD-L1-eGFP expressed in CHO-K1 cells allowed us, for the first time, to determine the affinity of the complex formed between PD-1 and its ligand PD-L1 during tumor escape. The protocol has a variety of potential applications for studying the interactions of proteins with small molecules.
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https://hal.univ-lille.fr/hal-03185893
Contributeur : Lilloa Université de Lille <>
Soumis le : mardi 30 mars 2021 - 16:53:29
Dernière modification le : jeudi 1 avril 2021 - 03:31:18

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Magnez Romain, Bryan Thiroux, Morgane Tardy, Bruno Quesnel, Xavier Thuru. Measurement of Protein-Protein Interactions through Microscale Thermophoresis (MST). Bio-protocol , Bio-protocol LCC, 2020, Bio-protocol, 10 (7), ⟨10.21769/bioprotoc.3574⟩. ⟨hal-03185893⟩

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