SUIT-024 O2 ce-pce D056

high-resolution terminology - matching measurements at high-resolution

SUIT-024 O2 ce-pce D056

Description

Abbreviation: ceGlc

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

SUIT number: D056_ce1;1Dig;1PM;2T;2D;3Omy;4Am

O2k-Application: O2

The SUIT-024 is designed to test the contamination of the sample with ATPases. When isolating mitochondria, contamination with other membranous organelles containing ATPases (e.g., plasma membrane and endoplasmic reticulum) can occur. In experiments with tissue homogenates and permeabilized cells, it is expected to have active ATPases in the respiration medium. When ATPases are active and residual endogenous adenylates are present in the sample, ATP can be consumed and converted to ADP, which will stimulate respiration. This may lead to an overestimation of LEAK respiration - L(n). Therefore, 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). This protocol can be used as a quality control of the mitochondrial preparation.

In the DatLab software, SUIT-024 DLP file is provided for ce-pce and the category N(PM). For using this protocol with other sample preparation or substrate/inhibitor combinations, a personalized DLPU can be created.

Communicated by Garcia-Souza LF (last update 2019-05-14)

Strengths and limitations

• This protocol allows to analyse the presence of ATPases in mitochondrial preparations and is useful as a quality control of the sample preparation.

+ Different combinations of substrates and inhibitors 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 

MitoPedia methods: Respirometry