Cells were grown overnight at 30 °C in YPD medium, and mitochondria were isolated by using a Yeast Mitochondria Isolation Kit (Sigma-Aldrich) according to the manufacturer’s instructions. Mitochondria (50 µg) were prepared to measure F1Fo-ATPase activity. The ATP hydrolysis reaction was initiated by adding 1 mM ATP and then phosphate release was monitored at an absorbance of 360 nm using an EnzChek Phosphate Assay Kit (Invitrogen) according to the manufacturer’s instructions. The average hydrolysis rates in a time period from 1 to 5 min after initiation were calculated and presented as the amount of phosphate (mmol) released per min.
Yeast mitochondria isolation kit
Manufactured by Merck Group
The Yeast Mitochondria Isolation Kit is a laboratory equipment designed to isolate and purify mitochondria from yeast samples. The kit provides a standardized and efficient method for extracting mitochondria, which are the energy-producing organelles within eukaryotic cells.
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2 protocols using yeast mitochondria isolation kit
1
Yeast Mitochondrial F1Fo-ATPase Activity
2
Mitochondria Isolation from Aspergillus
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Aspergillus nidulans protoplasts were applied to the “Yeast Mitochondria Isolation Kit” (SigmA) and mitochondria isolated as described in the manufacturer’s protocol using detergent lysis (1:200 dilution). In the first centrifugational step, at 600× g mitochondria were obtained in the supernatant (S1). In the second centrifugational step, at 6,500× g mitochondria were sedimented in the pellet (P2). Proteins were analyzed in a Western blot using anti‐GFP antibodies (Roche). For precipitation of the GFP fusion protein (GFP trap), the protein extracts were incubated with the anti‐GFP antibody and protein G agarose (Roche).
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