Reference: A: Protocol for coupling control assessment in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells) SUIT-006
SUIT number: D022_1SRot;2D;2c;(3Omy);4U;5Ama
SUIT-006 O2 mt D022 is a coupling control protocol for mitochondrial preparations (tissue homogenate, isolated mitochondria and permeabilized cells). Different coupling control states are assessed (L(n) - P - E) at succinate pathway control state. The use of rotenone, that inhibits complex I, provides a specific coupling control protocol for S-pathway, avoiding reverse electron transfer (RET).
In the DatLab software, the DLP file for SUIT-006 O2 mt D022 is provided without the addition of oligomycin. For using this protocol with oligomycin addition, a DLPU can be created, with the Omy event and mark. Using oligomycin in the preliminary assays with a not yet studied model is useful to determine whether in this kind of sample LEAK respiration in the absence of ADP (1SRot) may be overestimated due to the presence of residual and endogenous adenylates.
Communicated by Cardoso LH, Komlodi T, Huete-Ortega M, Iglesias-Gonzalez J, Doerrier C 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
- + The combination of succinate with rotenone, that inhibits complex I, provides the best coupling control protocol specific for S-pathway, avoiding reverse electron transfer (RET).
- + Reasonable duration of the experiment.
- + This protocol can be extended with the Complex IV module.
- - This protocol does not include internal ET-pathway control steps.
- - Careful washing is required after the experiment to avoid carry-over of uncoupler and inhibitors.
Compare SUIT protocols
|Krumschnabel 2014 Methods Enzymol||2014||Mouse||Nervous system|
MitoPedia methods: Respirometry