Dihydroethidium (Cat. # 50-850-563), FM-4-64 (Cat # T-3166), and Phen Green SK diacetate (Cat. # P-14313) was purchased from Thermo Fisher Scientific. Paraquat dichloride (Cat # 856177-1G) was purchased from Sigma-Aldrich. Yeast nitrogen base and SC dropout mixtures were purchased from Sunrise Science Products. The Yeast Mitochondria Isolation kit was purchased from Bio Vision (Cat. # K259-50). Rabbit polyclonal antibodies against GAPDH (Cat. # 89348-232) and GFP (Cat. # 89362-978) were purchased from VWR. Rabbit polyclonal antibody against PGK1 (Cat. #PA528612) and mouse monoclonal antibody against Porin (Cat. #459500) were purchased from Thermo Fisher and Invitrogen, respectively. A previously described rabbit polyclonal antibody against Sod1 was obtained from the laboratory of Valeria Culotta (Johns Hopkins University) [50] (link).
Yeast mitochondria isolation kit
Manufactured by Abcam
Sourced in United States
The Yeast Mitochondria Isolation Kit is a tool designed to isolate mitochondria from yeast cells. It provides a simple and efficient method to extract and purify mitochondria, which are the organelles responsible for cellular respiration and energy production in yeast.
Automatically generated - may contain errors
4 protocols using yeast mitochondria isolation kit
1
Yeast Mitochondria Isolation and Analysis
2
Mitochondrial ATP and ATPase Assay
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For the determination of the ATP content and ATPase activity, mitochondria were isolated using a yeast mitochondria isolation kit (Biovision, San Francisco, CA, USA), and the content of protein was determined using a BCA protein assay kit (Solarbio, Beijing, China). Then, the ATP content and ATPase activity of the mitochondria were determined using an ATP Colorimetric/Fluorometric assay kit and an ATPase/GTPase activity assay kit, respectively. All operations were performed according to the manufacturer’s instruction, and each experiment was performed at least three times.
3
Isolation and Analysis of Yeast Mitochondria
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Mitochondria were isolated using the Yeast Mitochondria Isolation Kit (Bio Vision) according to the manufacturer's specifications. For this purpose, sod1∆ cells expressing an empty vector (pRS415), SOD1 (pRS415-SOD1) or IMS localized SCO2-SOD1 (pRS415-SCO2-SOD1) were grown in 20 mL, SC-2% glucose cultures to a density of OD600 nm = 1.0. 4 × 108 cells of each strain were harvested and washed in ice-cold ultra pure water prior to fractionation. After fractionation, volumes corresponding to 2.5% of the whole cell extract and cytosolic fractions, and 10% of the mitochondrial fraction were assessed for Pgk1, Sod1, and Porin expression by SDS-PAGE and immunoblotting. Pgk1 and Sod1 were probed with anti-PGK1 (1:1000) and anti-SOD1 (1:5000) polyclonal antibodies and detected using a goat anti-rabbit secondary antibody conjugated to a 680 nm emitting fluorophore (Biotium). Porin was probed with an anti-Porin (1:5000) monoclonal antibody and detected using a goat anti-mouse secondary antibody conjugated to a 797 nm emitting fluorophore (Thermo Fisher).
4
Isolation and Purification of Yeast Mitochondria
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Mitochondria were isolated from yeast cells by the yeast mitochondria isolation kit (BioVision, CA) according to the manufacturer's instructions. Although the resulting samples are enriched in mitochondria, they may contain other organelles such as the endoplasmic reticulum, Golgi complex, and vacuoles. To obtain pure mitochondria, this crude mitochondrial fraction was subjected to further fractionation as described [33 ]. Briefly, a sucrose gradient was constructed by multiple layers extending from 60% sucrose to 15% sucrose in EM buffer (10 mM MOPS/KOH (pH 7.2), 1 mM EDTA) in a Beckman Ultra-Clear centrifuge tube (Beckman Coulter Life Sciences). Three ml of the crude mitochondrial fraction in SEM buffer (10 mM MOPS/KOH, pH 7.2, 250 mM sucrose, and 1 mM EDTA) was placed on the top of 15% (w/v) sucrose and centrifuged in a Beckman SW44 Ti swinging-bucket rotor for 1 h at 134,000g at 4°C. The intact mitochondria were obtained from a brown band at the 60%/32% sucrose interface, and purified mitochondria were used for sphingolipid analyses, mitochondrial membrane potential assays, and mitochondrial fusion assays.
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