Reference: A is to analyse the coupling control in NADH-pathway in mitochondrial prepartions (isolated mitochondria and tissue homogenate) - SUIT-006
SUIT number: D047_1PM;2D;2c;3Omy;4U;5Ama
SUIT-006 O2 mt D047 is a coupling control protocol for mitochondrial preparations (tissue homogenate, isolated mitochondria and cells permeabilized before addition to the O2k chamber). Different coupling control states are assessed (L(n) - P - L(Omy) - E) at N(PM) pathway control state. The use of oligomycin is optional, however, it provides important information when residual and endogenous adenylates are present (which may happen if ATPases are active in the sample). This situation may lead to overestimated LEAK respiration measured in the absence of adenylates - L(n). Therefore, oligomycin can be used to verify whether this occurs and obtain the LEAK state appropriately.
Communicated by Cardoso LH, Iglesias-Gonzalez J, Komlodi T and Gnaiger E (last update 2019-04-02)
Steps and respiratory states
|Step||State||Pathway||Q-junction|| Comment - Events (E) and Marks (M)
- » Selected keywords in SUIT protocols
- Pathway control
Strengths and limitations
- This protocol is indicated when the aim is to analyse coupling control in mitochondrial preparations in NADH-pathway using PM as substrates.
- + Reasonable duration of the experiment.
- + This protocol can be extended with the Complex IV module.
- - This protocol analyses the coupling control in NADH-pathway (with PM), however, it is less specific than a similar protocol containing the complex II inhibitor malonic acid, that would allow to analyse the NADH-pathway without any contribution of the S-pathway.
- - Careful washing is required after the experiment to avoid carry-over of uncoupler and inhibitors, if used.
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MitoPedia methods: Respirometry