Difference between revisions of "Mitochondrial membrane potential"
(Created page with "{{MitoPedia |abbr=mt-membrane potantial }} {{MitoPedia methods}} {{MitoPedia topics}} This page is a guide to various wiki.oroboros.at pages that complement [http://www.oroboros....") |
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Practical difficulties in applying the method to permeabilized fibers are discussed in [[Mitochondrial membrane potential of permeabilized fibers]]. Some tips for [[Cleaning the TPP+ electrodes]]Some important performance parameters of the TPP electrode are summarized on this [http://www.oroboros.at/fileadmin/user_upload/MiP2010/MiP2010_Sumbalova_poster1.pdf poster]. In any way we hope you will join us for one of our [[TPP special interest group during an International Oxygraph Course]]. | Practical difficulties in applying the method to permeabilized fibers are discussed in [[Mitochondrial membrane potential of permeabilized fibers]]. Some tips for [[Cleaning the TPP+ electrodes]]Some important performance parameters of the TPP electrode are summarized on this [http://www.oroboros.at/fileadmin/user_upload/MiP2010/MiP2010_Sumbalova_poster1.pdf poster]. In any way we hope you will join us for one of our [[TPP special interest group during an International Oxygraph Course]]. | ||
== Mitochondrial membrane potential and anoxia == | |||
=== Question === | |||
Can I use anoxia as a reference state for zero (minimum) mt-membrane potential in isolated mitochondria? I want to run experiments in series without loosing the time for washing out inhibitors or uncouplers. The protocol includes substrates and ADP. | |||
=== Answer === | |||
Anoxia should provide a good reference value for minimum mt-membrane potential. However, you should carry out a test experiment: After reaching anoxia, add oligomycin as a test for the possibility that ATPsynthase acts as a ATPase and thus maintains a mt-membrane potential in reversed mode of operation. Then titrate uncoupler (FCCP) to collaps the mt-membrane potential under anoxia. | |||
Careful: Ethanol as a carrier for oligomycin and FCCP exerts a chemical side effect on the TPP+ signal, which has to be evaluated in a separate control experiment and subtracted from the experimental trace. | |||
=== See also === | |||
[[Mitochondrial membrane potential]] | |||
[[ISE]] | |||
Revision as of 14:01, 5 October 2012
- high-resolution terminology - matching measurements at high-resolution
Mitochondrial membrane potential
Description
Abbreviation: mt-membrane potantial
This page is a guide to various wiki.oroboros.at pages that complement [1] with up-to-date information and discussions. The general manual for the OROBOROS Ion Selective Electrode system (ISE System) is in [MiPNet15.03]. While working with the potentiometric (pX) channel of the O2k, the general guidelines for avoiding damage to the oxygraph by ESD should be followed.
A rudimentary protocol for measuring mitochondrial membrane potential (MiPNet14.05), its mathematical appendix and the most up-to-date spreadsheet templates, DatLab templates, and DatLab demo files, can be found here.
Before measuring membrane potential via TPP+ is even started, the TPP+ inhibitory effect in the studied system should be explored.How to get from the measured TPP+ concentration to the mitochondrial membrane potential: Calculation of mitochondrial membrane potential from measurements with a TPP electrode. This covers also the influence of Unspecific binding.
Practical difficulties in applying the method to permeabilized fibers are discussed in Mitochondrial membrane potential of permeabilized fibers. Some tips for Cleaning the TPP+ electrodesSome important performance parameters of the TPP electrode are summarized on this poster. In any way we hope you will join us for one of our TPP special interest group during an International Oxygraph Course.
Mitochondrial membrane potential and anoxia
Question
Can I use anoxia as a reference state for zero (minimum) mt-membrane potential in isolated mitochondria? I want to run experiments in series without loosing the time for washing out inhibitors or uncouplers. The protocol includes substrates and ADP.
Answer
Anoxia should provide a good reference value for minimum mt-membrane potential. However, you should carry out a test experiment: After reaching anoxia, add oligomycin as a test for the possibility that ATPsynthase acts as a ATPase and thus maintains a mt-membrane potential in reversed mode of operation. Then titrate uncoupler (FCCP) to collaps the mt-membrane potential under anoxia.
Careful: Ethanol as a carrier for oligomycin and FCCP exerts a chemical side effect on the TPP+ signal, which has to be evaluated in a separate control experiment and subtracted from the experimental trace.
