SUIT-026 O2 mt D063

From Bioblast
Jump to: navigation, search
Bioblasts - Richard Altmann and MiPArt by Odra Noel
MitoPedia         Terms and abbreviations         Concepts and methods         MitoPedia: SUIT         MiP and biochemistry         Preprints and history



MitoPedia

SUIT-026 O2 mt D063

Description

1S;2Rot;3D;3c;4Ama.png

Abbreviation: RET (respiratory control) of SUIT-026 AmR mt D064

Reference: B - SUIT-026 - Protocol for the respiratory control of SUIT-026 AmR mt D064

SUIT number: D063_1S;2Rot;3D;3c;4Ama

O2k-Application: O2


SUIT-026 O2 mt D063 has been designed to serve as a respiratory control of the SUIT-026 AmR mt D064 (protocol to study the RET-initiated ROS production in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells which are already permeabilized when they are added to the chamber).

Communicated by  Komlodi T, Iglesias-Gonzalez J and Gnaiger E (last update 2019-09-24)


Representative traces

SUIT-026 O2 mt D063 O2 trace.png

MitoPedia: SUIT

Steps and respiratory states

1S;2Rot;3D;4Ama.png


Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1S SL(n) S CII 1S
2Rot SL(n) S CII 1S;2Rot
3D SP S CII 1S;2Rot;3D
3c SP S CII 1S;2Rot;3D;3c
  • Addition of cytochrome c yields a test for integrity of the mtOM. Stimulation by added cytochrome c would indicate an injury of the mtOM and limitation of respiration in the preceding state without added c due to loss of cytochrome c. Typically, cytochrome c is added immediately after the earliest ADP-activation step (P, OXPHOS capacity with saturating [ADP]).
  • Succinate, S ( type S-pathway to Q).
  • OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.
4Ama ROX 1S;2Rot;3D;3c;4Ama
  • Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated after addition of antimycin A (inhibitor of CIII). Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration (mt).


Questions.jpg


Click to expand or collaps

Strengths and limitations

  • This protocol serves as a respiratory control (since fluorescence dyes may inhibit components of the ET system) and quality control for the sample for SUIT-026 AmR mt D064.
+ Short protocol.
+ The addition of cytochrome c to assess the mt outer membrane integrity provides a quality control of the sample, which cannot be performed in the protocol with Amplex UltraRed.
- This protocol does not provide information about all the coupling control states (LEAK, OXPHOS and ET). However, it is possible to create a DLPU by additing an Event&Mark for the uncoupler titration (4U) after ADP (3D) to obtain ET-state.


Compare SUIT protocols


Chemicals and syringes

Step Chemical(s) and link(s) Comments
1S Succinate (S)
2Rot Rotenone (Rot)
3D ADP (D)
3c Cytochrome c (c)
4Ama Antimycin A (Ama)
Suggested stock concentrations are shown in the specific DL-Protocol.


References

MitoPedia concepts: SUIT protocol, SUIT B, Find 


MitoPedia methods: Fluorometry