Rat heart mitochondria (RHM) were isolated as described previously.20 (link) Briefly, freshly excised rat hearts were homogenized in STEB buffer [250 mM sucrose, 5 mM Tris, 1 mM EGTA, 0.1% (w/v) bovine serum albumin (BSA), pH 7.4] using a Dounce homogenizer. Homogenates were centrifuged (3000 × g, 5 min, 4°C) to pellet nuclei and unbroken cells. The resulting supernatant was then centrifuged (10 000 × g, 10 min, 4°C) to pellet mitochondria. Pelleted mitochondria were resuspended in STEB buffer and recentrifuged (10 000 × g, 10 min, 4°C). The pelleted mitochondria were resuspended in STEB buffer with the BSA omitted (400 µL/heart) and the mitochondrial protein quantified by bicinchoninic (BCA) assay (Thermo Fisher Scientific).
Prag H.A., Gruszczyk A.V., Huang M.M., Beach T.E., Young T., Tronci L., Nikitopoulou E., Mulvey J.F., Ascione R., Hadjihambi A., Shattock M.J., Pellerin L., Saeb-Parsy K., Frezza C., James A.M., Krieg T., Murphy M.P, & Aksentijević D. (2020). Mechanism of succinate efflux upon reperfusion of the ischaemic heart. Cardiovascular Research, 117(4), 1188-1201.
Assay Bovine serum albumin BufferCells EgtaHeart Heart mitochondriaMitochondria Mitochondrial protein NucleiSucrose Tris
Corresponding Organization : Queen Mary University of London
Other organizations :
University of Cambridge, Addenbrooke's Hospital, MRC Mitochondrial Biology Unit, MRC Cancer Unit, Hutchison/MRC Research Centre, University of Bristol, Bristol Royal Infirmary, University of Lausanne, King's College London, St Thomas' Hospital, British Heart Foundation, Université de Bordeaux, Centre de Résonance Magnétique des Systèmes Biologiques, Centre National de la Recherche Scientifique, Université de Poitiers, Inserm
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