Difference between revisions of "TMPD"
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:::: <span style="color:#8B008B"> '''Caution:''' Light sensitive (store solution in a dark glass vial). Take care to minimize light and temperature exposure during use!</span> | :::: <span style="color:#8B008B"> '''Caution:''' Light sensitive (store solution in a dark glass vial). Take care to minimize light and temperature exposure during use!</span> | ||
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::::* Titration volume: 5 µl using a 25 µl syringe. | ::::* Titration volume: 5 µl using a 25 µl syringe. | ||
::::* Final conc. in 2 ml O2k-chamber: 0.5 mM. | ::::* Final conc. in 2 ml O2k-chamber: 0.5 mM. | ||
:::: 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. Electrons flow from TMPD to cytochrome ''c''. | |||
:::: For preparation and titration please visit [[Ascorbate]] | |||
Revision as of 14:23, 14 January 2019
- 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
MitoPedia topics:
Substrate and metabolite
Application in HRFR
Complex IV assay
- For the Complex IV assay TMPD is required at 0.5 mM final concentration added from a 200 mM stock.
Preparation of 200 mM stock
- TMPD (C10H16N2*2HCl): Sigma T 3134, 5 g, store at RT; FW = 237.2.
- Weigh 47.4 mg TMPD
- Dissolve in 1 ml H2O
- Divide into 0.2 ml portions
- Protect from light!
- Store frozen at -20 °C
- Caution: Light sensitive (store solution in a dark glass vial). Take care to minimize light and temperature exposure during use!
- Oxygraph-2k Manual Titrations: MiPNet09.12
- Titration volume: 5 µl using a 25 µl syringe.
- Final conc. in 2 ml O2k-chamber: 0.5 mM.
- 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. Electrons flow from TMPD to cytochrome c.
- For preparation and titration please visit Ascorbate
