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Tissue was homogenised in a buffer (20 mM Tri-HCl (pH 8.0), 37 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% Triton X-100). The homogenate was centrifuged (10,000 g, 4 °C 5 mins) and 20 µl of the supernatant was added to 500 µl of assay buffer (30 °C) (100 mM KCl, 20 mM HEPES, 0.6 mM 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) pH 7.5). The reaction was started by addition of 2.5 mM palmitoyl-CoA and absorbance monitored for 5–10 minutes at 412 nm. An extinction coefficient of 13,600 M−1 cm−1 was used to calculate the thioesterase activity26.
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