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Difference between revisions of "Digitonin"

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Revision as of 11:55, 8 February 2016


high-resolution terminology - matching measurements at high-resolution


Digitonin

Description

Digitonin is a mild detergent that permeabilizes plasma membranes selectively due to their high cholesterol content, whereas mt-membranes with lower cholesterol content are affected only at higher concentrations. Digitonin is a natural product and thus the effective concentration has to be determined by titrations for every batch. The optimum effective digitonin concentrations for complete plasma membrane permeabilization of cultured cells can be determined directly in a respirometric protocol.

Abbreviation: Dig

Reference: Pesta 2012 Methods Mol Biol, MiPNet09.12 O2k-Titrations






MitoPedia topics: Permeabilization agent 

Application in HRR

Dig: Digitonin (C56H92O29); Sigma D 5628, 1 g, store at RT; FW = 1229.31


Caution: Toxic!


Preparation of 8.1 mM stock solution (dissolved in DMSO):

1. Weigh 10 mg of digitonin.
2) Dissolve in 1 ml DMSO.
3) Store at -20 ยฐC.


O2k Manual Titrations: MiPNet09.12 O2k-Titrations

  • Titration volume: 1 ยตl using a 10 ยตl syringe.
  • Final conc. in 2 ml O2k-chamber: 4.1 ยตM (typical optimum concentration for selective plasma membrane permeabilization).
  • Respirometric test of optimum digitonin concentration (Pesta 2012 Methods Mol Biol)
  1. Incubate cells in the O2k in mt-respiration medium (MiR05 or MiR06) and measure ROUTINE respiration.
  2. Titrate succinate (S, 10 mM) and ADP (D, 2 to 5 mM). Most intact cells are impermeable for S and D.
  3. Titrate rotenone (0.1 ยตM). CI inhibition in intact cells inhibits the TCA cycle such that succinate is not produced, hence a state close to minimum residual oxygen consumption (ROX) is obtained.
  4. Titrate digitonin using the Titration-Injection microPump (TIP2k) or manual titrations, to observe stepwise stimulation of respiration by increasing availability of S and D to the mitochondria, until a plateaux of maximum respiraiton is reached.

Examples are show here: Permeabilizing cells