Human recombinant PPARδ protein was expressed by transforming pET28a plasmid (custom cloning core facility at Emory University) into One Shot BL21 Star (DE3) cells (C601003, Thermo Fisher Scientific). Cells were lysed, and the recombinant protein was purified and concentrated according to standard protocols, as described previously.50 (link) We excluded reducing agents from all buffers.
The resulting recombinant PPARδ was incubated with 15d-PGJ2-Biotin (10141; Cayman Chemical) for 1 h, which was followed by 30-min incubation with or without DTT (10 or 100 mM). Cys-alkylation of the samples was then analyzed on an SDS-PAGE gel under non-reducing conditions, followed by Western blotting to detect 15d-PGJ2-Biotin (IRDye 680 streptavidin) and PPARδ (IRDye 800). The infrared signal was detected with an Odyssey Infrared Imager.
The resulting recombinant PPARδ was incubated with 15d-PGJ2-Biotin (10141; Cayman Chemical) for 1 h, which was followed by 30-min incubation with or without DTT (10 or 100 mM). Cys-alkylation of the samples was then analyzed on an SDS-PAGE gel under non-reducing conditions, followed by Western blotting to detect 15d-PGJ2-Biotin (IRDye 680 streptavidin) and PPARδ (IRDye 800). The infrared signal was detected with an Odyssey Infrared Imager.
