Difference between revisions of "Cytochrome c"
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== Application in [[ | == Application in [[HRR]]: storage and stock solution of ''c'' == | ||
:::: '''''c'': Cytochrome ''c'' ''' (from equine heart), Sigma C 7752, 50 mg, store at -20 °C; FW = 12384 Da. | :::: '''''c'': Cytochrome ''c'' ''' (from equine heart), Sigma C 7752, 50 mg, store at -20 °C; FW = 12384 Da. | ||
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::::# Weigh 50 mg cytochrome ''c'' into a small glass beaker. Difficult to weigh, since the powder is electromagnetic. | ::::# Weigh 50 mg cytochrome ''c'' into a small glass beaker. Difficult to weigh, since the powder is electromagnetic. | ||
::::# Add 1 | ::::# Add 1 mL H<sub>2</sub>O; ''c'' dissolves easily. | ||
::::# Divide into 0.2 | ::::# Divide into 0.2 mL portions. | ||
::::# Store at -20 °C. | ::::# Store at -20 °C. | ||
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::: '''O2k manual titrations:''' [[MiPNet09.12 O2k-Titrations]] | ::: '''O2k manual titrations:''' [[MiPNet09.12 O2k-Titrations]] | ||
::::* Titration volume: 5 | ::::* Titration volume: 5 µL using a 25 µL syringe (2 mL O2k-chamber). | ||
::::* Final concentration: 10 µM. | ::::* Final concentration: 10 µM. | ||
Revision as of 12:59, 17 March 2019
- high-resolution terminology - matching measurements at high-resolution
Cytochrome c
Description
Cytochrome c is a component of the Electron transfer-pathway (ET-pathway) in mitochondria. It is a small heme protein loosely associated with the outer side of the inner mitochondrial membrane. The heme group of cytochrome c transfers electrons from Complex III to Complex IV. The release of cytochrome c into the cytoplasm is associated with apoptosis.
Abbreviation: c
Reference: MiPNet09.12; Gnaiger 2002 Biochem Soc Trans
MitoPedia topics: Substrate and metabolite
Application in HRR: storage and stock solution of c
- c: Cytochrome c (from equine heart), Sigma C 7752, 50 mg, store at -20 °C; FW = 12384 Da.
- Preparation of 4 mM cytochrome c solution (dissolved in H2O):
- Weigh 50 mg cytochrome c into a small glass beaker. Difficult to weigh, since the powder is electromagnetic.
- Add 1 mL H2O; c dissolves easily.
- Divide into 0.2 mL portions.
- Store at -20 °C.
- Caution: Chemicals stored in the fridge or freezer should be allowed to reach room temperature before opening.
- O2k manual titrations: MiPNet09.12 O2k-Titrations
- Titration volume: 5 µL using a 25 µL syringe (2 mL O2k-chamber).
- Final concentration: 10 µM.
Performance and data analysis
Mitochondrial preparation is one of the key steps for the quality of our mitochondrial respiration assessments. During the isolation of the organelles or the permeabilization of the cells/fibers, we must ensure the integrity of the outer membrane. However, some of the technique used like the disruption of the tissue for mitochondrial isolation or the permeabilization of the cells with digitonin could compromise this integrity. For this reason, the addition of the cytochrome c is routinely used to test of the outer membrane permeability and to evaluate the quality of our mitochondrial preparation. Therefore, the addition of cytochrome c will stimulate the respiration if the outer mitochondrial membrane has been altered.
It is common to find stimulation in the respiration, and the percentage of increase should be determined empirically because it is sample specific. For example, in the case of the mouse brain isolated mitochondria it is well characterized that the cytochrome c should not stimulate the OXPHOS respiration more than 20%. However, in other preparations such as the muscle fiber from mice, it has been described a 30% increase in the respiration. If there are no data available in the literature for our sample, we need to accumulate enough data and look for consistency.
- For further information see: » Cytochrome c control factor
