Abbreviation: RP1-short pfi
SUIT number: D010_1PM;2D;2c;3U;4S;5Rot;6Ama;7AsTm;8Azd
The SUIT-004 O2 pfi D010 protocol provides information of the linear coupling control (L- P- E) with NADH linked-substrates (PM). Moreover, the pathway control in ET state (N, NS and S) can be evaluated by using this SUIT protocol. The SUIT-004 O2 pfi D010 can be extended with the CIV assay module. Permeabilized muscle fibers are sensitive to oxygen supply due to limited diffusion of oxygen to the fiber bundle core. To counteract this limitation, hyperoxic conditions (400-250 µM O2) must be employed. To set the optimal oxygen concentration in the O2k-Chamber, see Setting the oxygen concentration.
Communicated by Doerrier C, Iglesias-Gonzalez J and Gnaiger E (last update 2019-06-05)
Steps and respiratory states
|Step||State||Pathway||Q-junction||Comment - Events (E) and Marks (M)|
|Step||Respiratory state||Pathway control||ET-Complex entry into Q-junction||Comment|
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- Pathway control
Strengths and limitations
- A succinate concentration of >10 mM may be required for saturating SE capacity.
- CIV assessment is skipped if a prolonged protocol is not practical due to time limitation.
- RP1-6Ama may be skipped since correction of the initial states by ROX measured in RP1-6 Ama may represent an over-correction, and inhibition takes a very long time in several cases (Pesta 2012 Methods Mol Biol).
- + Linear coupling control (L- P- E) with NADPH-linked substrate substrates (PM).
- + Pathway control in ET state (N, NS, S).
- + This protocol can be extended with the Complex IV module.
- + Short experimental time.
- - The substrate combination for maximum ET capacity (FNSGP) is not obtained in SUIT-004 in favor of reducing the time consumed during the experiment.
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MitoPedia methods: Respirometry