SUIT protocol library

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



MitoPedia

SUIT protocol library

Description

The Substrate-uncoupler-inhibitor titration (SUIT) protocol library contains classes of SUIT protocols with coupling and substrate control defined for mitochondrial preparations.

Abbreviation: SUITs

Reference: MitoPedia: SUIT


MitoPedia concepts: MiP concept, SUIT concept 






This page is in preparation (intern wiki: [1])

Strategy

  1. Select a pathway control state.
  2. Select a control pattern.
  3. Define substrates for each pathway state.
  4. Define coupling states.


Definition of states

Experimental conditions

  • Temp
  • O2
  • Medium
  • Oxygen level
  • ADP kinetics
  • Substrate kinetics

O2k configuration

  • Amp: Amplex Red, TMRM, Ca Green, ...
  • Pot
  • Spectrophotometry


Define substrates

Substrate control states are defined for each pathway control state (CI-linked, CII-linked, CI&II-linked) is defined by a specific substrate or substrate combination. These substrates may be added in a single step, or in multiple steps for analyis of the effect of different substrate combinations within a pathway control state.
  • CI: PM, GM, PGM, M, ..
  • CII: S(Rot), S, SM, ..
  • CI&II: PS, GS, PMS, GMS, PGMS, ..
  • CIV: AsTM
  • FAO: Oct, Pal, ...
  • CGpDH:
  • c

Define coupling states

The coupling control states require a complementary definition of an ETS-competent pathway control state.

Coupling states: LEAK OXPHOS ETS


Define residual oxygen consumption

  • ROX: Rot&Ama, Rot&Mna&Ama, Myx, Cn, Az, ..

ROX


Categories of SUIT protocols

» Categories of SUIT protocols
» Talk:SUIT protocol library

SUIT protocol patterns

» SUIT protocol pattern


SUIT library

Work in progress
» MitoPedia: SUIT

CI + CI&II + CII diametral

Respiratory states: CIL -> CIP -> CI&IIP -> CI&IIE -> CIIE -> ROX
  • GM+D+S+U+Rot Innsbruck-Copenhagen protocol: GM+D+c+S+U+Rot ::Cyt c: added after ADP; cyt c added after S; ADP conc: _ _; ROX: Ama (Boushel 2007 Diabetologia)
  • PMG+D+S+U+Rot Brain protocol: PMG+D+c+S+U+Rot::ADP conc: in mouse brain homogenate a final conc. of 2 mM ADP in the OXPHOS state (CI&II) is saturating (Sumbalova 2011 Abstract Bordeaux); ROX: Mna&Ama; Myx


CI + CI + CI&II + CII diamteral

  • GM+D+P+S+U+Rot Innsbruck-Liege protocol 1: GM+D+c+P+S+U+Rot::Cytochrome c: early addition of c ensures comparability of all states in case of any c effect.
  • PM+D+G+S+U+Rot Innsbruck-Liege protocol 2: PM+D+c+G+S+U+Rot


FAO + CI + CI&II + CII diametral

Respiratory states: FAOL -> FAOP -> CI&FAOP -> CI&II&FAOP -> CI&II&FAOE -> CIIE -> ROX


Respiratory states: FAOL -> FAOP -> CI&FAOP -> CI&II&FAOP -> CIIP -> CIIL -> CIIE -> ROX
  • OctM+D+G+S+Rot+Omy+U Greenland-Monte Rosa protocol: OctM+D+G+S+Rot+Omy+U+c::Cyt c: added after U. The absence of a significant c-effect shows the integrity of the outer mitochondrial membrane after fibre preparation and also shows the preservation of integrity over a 90-100 min incubation in the O2k (MiR05); Oxygen: oxygen limitation of flux was preventd by maintaining oxygen levels in the respirometer above air saturation (Pesta 2012 Methods Mol Biol); ROX: Mna+Ama


References and notes