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Updated: Nov 9, 2025

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay
Published on: January 7, 2019
Direct and specific binding of cholesterol to the mitochondrial translocator protein (TSPO) using PhotoClick
Elias Georges1,2, Chantal Sottas2, Yuchang Li2
1Institute of Parasitology, McGill University, Montreal, Quebec H9X1C0, Canada.
Abstract:
The translocator protein (TSPO) is a five-helix transmembrane protein localized to the outer mitochondria membrane. Radioligand binding assays and chemical crosslinking showed TSPO to be a high affinity cholesterol-binding protein. In this report, we show that TSPO in mitochondrial fractions from MA-10 mouse tumour Leydig cells can interact directly and competitively with the clickable photoreactive cholesterol analogue. PhotoClick cholesterol showed saturable photoaffinity labelling of TSPO that could be specifically immunoprecipitated with anti-TSPO antibody, following the click reaction with the fluorescent-azide probe, tetramethylrhodamine (TAMRA)-azide. Moreover, excess cholesterol reduced the photolabelling of both total mitochondrial proteins and TSPO. Together, the results of this study demonstrated direct binding of PhotoClick cholesterol to TSPO and that this interaction occurs at physiologically relevant site(s).
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