SUIT-024

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SUIT-024

Description

Abbreviation: ce

Reference: A: Determination of the presence of ATPases in mitochondrial preparations.

O2k-Application: O2

SUIT-category: N

SUIT protocol pattern: diametral ce1;1Dig;1PM;2T;2D;3Omy;4Ama

The SUIT-024 is designed designed to test the contamination of ATPases in the sample. When isolating mitochondria, contamination with other membranous organelles with ATPases (e.g., plasma membrane and endoplasmic reticulum) can occur. When ATPases are active and residual endogenous adenylates are present in the sample, ATP can be consumed and converted to ADP, which may lead to an overestimation of LEAK respiration. In experiments with tissue homogenates and permeabilized cells, it is expected to have active ATPases in the respiration medium. In brain tissue, synaptosomes are particularly difficult to be separated from mitochondria, making this protocol a good quality control of the imt fraction. If the samples are contaminated with ATPases/adenylates, it might be necessary to assess LEAK respiration in the presence of ATPases inhibitors or oligomycin - L(Omy).

Communicated by Garcia-Souza LF and Cardoso LH (last update 2019-05-07)

Specific SUIT protocols

SUIT-024 O2 ce D056

• SUIT-024 O2 ce D056 for intact cells (ce)

Strengths and limitations

• This protocol allows to analyse the presence of ATPases in mitochondrial preparations.

+ Different combinations of substrates and inhibitors (X) can be used depending on the specific aims, e.g.: PGM, PM, GM, MnaPM or other combinations for N-pathway control state; S or RotS for S-pathway control state.

+ LEAK respiration overestimation is prevented in the presence of Oligomycin.

+ Reasonable duration of the experiment.

- This protocol does not include internal ET-pathway control steps.

- CIV activity is not measured, to save experimental time.

- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.

References

MitoPedia concepts: MiP concept, SUIT protocol, Recommended 

MitoPedia methods: Respirometry