TMPD: Difference between revisions
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:::: During the respirometry experiment it is important to add [[Ascorbate]] into the chamber shortly before TMPD to avoid uncontrolled autoxidation. Ascorbate maintains TMPD in a reduced state, and electrons flow from TMPD to cytochrome ''c''. For preparation and titration please visit [[Ascorbate]]. | :::: During the respirometry experiment it is important to add [[Ascorbate]] into the chamber shortly before TMPD to avoid uncontrolled autoxidation. Ascorbate maintains TMPD in a reduced state, and electrons flow from TMPD to cytochrome ''c''. For preparation and titration please visit [[Ascorbate]]. | ||
:::: 2021-03: The preparation instructions were updated to take the volume of the solute (Gp) into account (see: [[Volume_of_the_solute]]). | |||
::::''' Preparation of 200 mM stock solution''' (dissolved in H<sub>2</sub>O) for '''2-mL O2k-chamber''': | |||
::::''' Preparation of 200 mM stock solution''' (1 mL, dissolved in H<sub>2</sub>O) for '''2-mL O2k-chamber''': | |||
::::# Weigh 47.4 mg TMPD | ::::# Weigh 47.4 mg TMPD | ||
::::# Dissolve in | ::::# Dissolve in 970 µL H<sub>2</sub>O | ||
::::# Divide into 0.2 mL portions | ::::# Divide into 0.2 mL portions | ||
::::# Protect from light! | ::::# Protect from light! | ||
| Line 30: | Line 32: | ||
::::# Weigh 59,25 mg TMPD | ::::# Weigh 59,25 mg TMPD | ||
::::# Dissolve in | ::::# Dissolve in 962 µL H<sub>2</sub>O | ||
::::# Divide into 0.2 mL portions | ::::# Divide into 0.2 mL portions | ||
::::# Protect from light! | ::::# Protect from light! | ||
Revision as of 11:24, 9 March 2021
- high-resolution terminology - matching measurements at high-resolution
TMPD
Description
N,N,N',N'-Tetramethyl-p-phenylenediamine dihydrochloride, TMPD, is applied as an artificial substrate for reducing cytochrome c in the respirometric assay for cytochrome c oxidase (CIV) activity. It is maintained in a reduced state by ascorbate and undergoes autoxidation as a function of oxygen pressure, TMPD, ascorbate and cytochrome c concentration.
Abbreviation: Tm
Reference: MiPNet06.06, MiPNet09.12
Application in HRR
- TMPD (C10H16N2*2HCl): Sigma T 3134, 5 g, store at RT; M = 237.2 g·mol-1.
- Caution: Light sensitive (store solution in a dark glass vial). Take care to minimize light and temperature exposure during use!
- Complex IV assay : For the Complex IV assay TMPD is required at 0.5 mM final concentration added from a 200 mM stock.
- TMPD (C10H16N2*2HCl): Sigma T 3134, 5 g, store at RT; M = 237.2 g·mol-1.
- 2021-03: The preparation instructions were updated to take the volume of the solute (Gp) into account (see: Volume_of_the_solute).
- Preparation of 200 mM stock solution (1 mL, dissolved in H2O) for 2-mL O2k-chamber:
- Weigh 47.4 mg TMPD
- Dissolve in 970 µL H2O
- Divide into 0.2 mL portions
- Protect from light!
- Store frozen at -20 °C
- Preparation of 200 mM stock solution (1 mL, dissolved in H2O) for 2-mL O2k-chamber:
- » O2k manual titrations MiPNet09.12 O2k-Titrations
- Titration volume (2-mL O2k-chamber): 5 µL using a 25 µL Hamilton syringe.
- Final concentration: 0.5 mM.
- Preparation of 250 mM stock solution (dissolved in H2O) for 0.5-mL O2k-chamber:
- Weigh 59,25 mg TMPD
- Dissolve in 962 µL H2O
- Divide into 0.2 mL portions
- Protect from light!
- Store frozen at -20 °C
- » O2k manual titrations MiPNet09.12 O2k-Titrations
- Titration volume (0.5-mL O2k-chamber): 1 µL using a 10 µL Hamilton syringe.
- Final concentration: 0.5 mM.
MitoPedia topics: Substrate and metabolite
