Difference between revisions of "Template:SUIT text D074"
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SUIT-031 Q ce-pce D074 is a protocol to investigate simultaneously oxygen flux and the redox state | SUIT-031 Q ce-pce D074 is a protocol to investigate simultaneously oxygen flux and the Q-redox state in the [[NADH Electron transfer-pathway state| N-]] and [[NS-pathway control state| NS-pathway]] and [[S-pathway control state]] in OXPHOS and in the [[S-pathway control state]] in the ET state in permeabilized cells. After permeabilization of the plasma membrane, [[coenzyme Q2]] mimetic is titrated since the naturally occurring oQ is trapped in the mitochondrial inner membrane. CoQ<sub>2</sub> reacts both with the mitochondrial complexes at the Q-binding site (CI, CII, and CIII) and with the detecting electrode of the [[Q-Sensor]]. Application of the lowest possible CoQ<sub>2</sub> concentration is recommended to avoid any side reactions on the ETS caused by the mimetics. Our study shows that 1 µM CoQ<sub>2</sub> was sufficient to detect the Q-redox change without an influence on respiration. <br> | ||
The addition of PM leads to a partial reduction of | The addition of PM leads to a partial reduction of CoQ<sub>2</sub> which is reflected in the increase of the Q-signal. ADP oxidizes CoQ<sub>2</sub>, reflected in the decrease of the Q-signa. Addition of S initiates NS-OXPHOS capacity and results in further reduction of CoQ<sub>2</sub> in the OXPHOS state. Rotenone via CI inhibition reduces O<sub>2</sub> flux to S-linked OXPHOS capacity and causes oxidation of CoQ<sub>2</sub>. The [[uncoupler]] CCCP oxidizes CoQ<sub>2</sub>, however, using mouse cardiac mitochondria (see figures) U did not influence either O<sub>2</sub> flux or the [[Q redox state]] which means that the respiration is not limited by the phosphorylation system in this sample type. <br> | ||
Anoxia was reached when the | Anoxia was reached when the mitochondria consumed the oxygen in the O2k-chambers. In the absence of O<sub>2</sub>, the ETS upstream of CIV is reduced and thus leads to the full reduction of CoQ<sub>2</sub>. This step is used as a reference step when calculating the [https://wiki.oroboros.at/index.php/Q_redox_state Q redox fraction]. At the end of the protocol, the CIII inhibitor antimycin A can be added to check its effect on the fully reduced CoQ<sub>2</sub> under anoxia. | ||
In the DatLab software, SUIT-031 DLP files are currently provided for different applications. For O<sub>2</sub> application with ce-pce, choose [[]], for mt, choose [[SUIT-031]]. For Q redox state detection with mt, choose [[SUIT-031 Q mt D072]].<br> | |||
How to measure the Q redox state, see: [[MiPNet24.12 NextGen-O2k: Q-Module]] | How to measure the Q redox state, see: [[MiPNet24.12 NextGen-O2k: Q-Module]] | ||
Latest revision as of 16:48, 25 November 2021
SUIT-031 Q ce-pce D074 is a protocol to investigate simultaneously oxygen flux and the Q-redox state in the N- and NS-pathway and S-pathway control state in OXPHOS and in the S-pathway control state in the ET state in permeabilized cells. After permeabilization of the plasma membrane, coenzyme Q2 mimetic is titrated since the naturally occurring oQ is trapped in the mitochondrial inner membrane. CoQ2 reacts both with the mitochondrial complexes at the Q-binding site (CI, CII, and CIII) and with the detecting electrode of the Q-Sensor. Application of the lowest possible CoQ2 concentration is recommended to avoid any side reactions on the ETS caused by the mimetics. Our study shows that 1 µM CoQ2 was sufficient to detect the Q-redox change without an influence on respiration.
The addition of PM leads to a partial reduction of CoQ2 which is reflected in the increase of the Q-signal. ADP oxidizes CoQ2, reflected in the decrease of the Q-signa. Addition of S initiates NS-OXPHOS capacity and results in further reduction of CoQ2 in the OXPHOS state. Rotenone via CI inhibition reduces O2 flux to S-linked OXPHOS capacity and causes oxidation of CoQ2. The uncoupler CCCP oxidizes CoQ2, however, using mouse cardiac mitochondria (see figures) U did not influence either O2 flux or the Q redox state which means that the respiration is not limited by the phosphorylation system in this sample type.
Anoxia was reached when the mitochondria consumed the oxygen in the O2k-chambers. In the absence of O2, the ETS upstream of CIV is reduced and thus leads to the full reduction of CoQ2. This step is used as a reference step when calculating the Q redox fraction. At the end of the protocol, the CIII inhibitor antimycin A can be added to check its effect on the fully reduced CoQ2 under anoxia.
In the DatLab software, SUIT-031 DLP files are currently provided for different applications. For O2 application with ce-pce, choose [[]], for mt, choose SUIT-031. For Q redox state detection with mt, choose SUIT-031 Q mt D072.
How to measure the Q redox state, see: MiPNet24.12 NextGen-O2k: Q-Module
