Membrane Protein Purification and Crystallization 2003
DOI: 10.1016/b978-012361776-7/50012-7
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Purification of the Cytochrome bc1 Complex from Yeast

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Cited by 9 publications
(10 citation statements)
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“…, respectively, which is comparable with published results (33,39,40). Even though the purification of the complexes utilized detergents, tightly bound lipids were previously found associated with highly purified preparations (41).…”
supporting
confidence: 83%
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“…, respectively, which is comparable with published results (33,39,40). Even though the purification of the complexes utilized detergents, tightly bound lipids were previously found associated with highly purified preparations (41).…”
supporting
confidence: 83%
“…Mitochondria (80 mg of protein) were resuspended in 10 ml of lysis buffer containing 2% dodecyl ␤-D-maltoside (DDM, Anatrace), 50 mM potassium acetate, 10% glycerol, 1:50 volume of protease inhibitor mixture for fungal and yeast cells (Sigma), 1.5 mM phenylmethylsulfonyl fluoride, and 30 mM HEPES-KOH, pH 7.4, for 90 min at 4°C. After centrifugation (145,000 ϫ g, 20 min), the supernatant was loaded on a DEAE CL-6B column (GE, 4 ml of resin) for CIII immobilization (33). The flowthrough was collected and saved for CIV purification (see below).…”
Section: Methodsmentioning
confidence: 99%
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“…(c and d) The activity of the cytochrome bc 1 complex (c) and the cytochrome c oxidase (d) was determined in submitochondrial particles prepared from wild-type, psd1 Δ, and psd1 Δ psd2 Δ yeast cells as described earlier 83,84 . Ubiquinol-dependent cytochrome c reduction was measured as described by Palsdottir and Hunte 84 using 3 μg protein (submitochondrial particles), 50 μM horse heart cytochrome c , and 80 μM decylubiquinol for 1 ml assay volume (40 mM potassium phosphate buffer, pH 7.4, 1 mM NaN 3 , and 0.05 % β- d -undecylmaltoside). Reduction of cytochrome c was monitored at 550 nm and the activity was calculated with an extinction coefficient of 19.4 mM − 1  cm − 1 .…”
Section: Figmentioning
confidence: 99%
“…Transformed cells were cultured at 30 °C in synthetic complete (SC) medium in the presence of 2% (w/v) glucose without uracil [45]. Cultures were diluted to an A 600 nm of 0.4 in SC medium containing 2% (w/v) galactose and growth was continued for 12 h. Cells were harvested by centrifugation and immediately used for the isolation of total membranes [46] or mitochondria [28,47]. Microsomes were separated from mitochondria by centrifugation of the resulting supernatant at 100 000 g for 45 min at 4 °C (Ti45, Beckman Coulter, Fullerton, CA, USA).…”
Section: Methodsmentioning
confidence: 99%