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Difference between revisions of "Digitonin"

From Bioblast
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|mitopedia topic=Permeabilization agent
|mitopedia topic=Permeabilization agent
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== Application in [[HRR]] ==
== Application in [[HRFR]] ==
::: '''Dig: Digitonin''' (C<sub>56</sub>H<sub>92</sub>O<sub>29</sub>); Sigma D 5628, 1 g, store at RT; FW = 1229.31
::: '''Dig: Digitonin''' (C<sub>56</sub>H<sub>92</sub>O<sub>29</sub>); Sigma D 5628, 1 g, store at RT; FW = 1229.31


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'''O2k Manual Titrations:'''  [[MiPNet09.12 O2k-Titrations]]
::: '''O2k Manual Titrations:'''  [[MiPNet09.12 O2k-Titrations]]


::* Titration volume: 1 µl using a 10 µl syringe.
::::* Titration volume: 1 µl using a 10 µl syringe.
::* Final conc. in 2 ml O2k-chamber: 4.1 µM (typical optimum concentration for selective plasma membrane permeabilization).
::::* Final conc. in 2 ml O2k-chamber: 4.1 µM (typical optimum concentration for selective plasma membrane permeabilization).
::* '''Respirometric test of optimum digitonin concentration''' ([[Pesta 2012 Methods Mol Biol]])
::::* '''Respirometric test of optimum digitonin concentration''' ([[Pesta 2012 Methods Mol Biol]])
::# Incubate cells in the O2k in mt-respiration medium (MiR05 or [[MiR06]]) and measure ROUTINE respiration.
::::# Incubate cells in the O2k in mt-respiration medium (MiR05 or [[MiR06]]) and measure ROUTINE respiration.
::# Titrate succinate (S, 10 mM) and ADP (D, 2 to 5 mM). Most intact cells are impermeable for S and D.
::::# Titrate succinate (S, 10 mM) and ADP (D, 2 to 5 mM). Most intact cells are impermeable for S and D.
::# Titrate rotenone (0.1 µM). C<sub>I</sub> 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.
::::# Titrate rotenone (0.1 µM). C<sub>I</sub> 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.
::# 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.
::::# 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: [[Talk:Digitonin#Examples_for_optimization_of_digitonin_for_permeabilizing_cells| Permeabilizing cells]]
::::* Examples: [[Talk:Digitonin#Examples_for_optimization_of_digitonin_for_permeabilizing_cells| Permeabilizing cells]]

Revision as of 10:29, 22 August 2017


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 HRFR

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


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. Divide into 0.1 ml portions
  4. Store at -20 °C.


O2k Manual Titrations: MiPNet09.12 O2k-Titrations
  • Titration volume: 1 µl using a 10 µl syringe.
  • Final conc. in 2 ml O2k-chamber: 4.1 µM (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 µM). 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.