Difference between revisions of "TMPD"
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<spanĀ style="color:#8B008B"> '''Caution:''' Light sensitive (storeĀ solution in a dark glass vial). Take care to minimize light andĀ temperature exposure during use!</span> | <spanĀ style="color:#8B008B"> '''Caution:''' Light sensitive (storeĀ solution in a dark glass vial). Take care to minimize light andĀ temperature exposure during use!</span> | ||
It isĀ important to addĀ [[Ascorbate]] before TMPDĀ to avoidĀ uncontrollableĀ autoxidation.Ā Ascorbate maintains TMPD in aĀ reducedĀ state. ElectronsĀ flow from TMPD to cytochrome c. | It isĀ important to addĀ [[Ascorbate]]shortly before TMPDĀ to avoidĀ uncontrollableĀ autoxidation.Ā Ascorbate maintains TMPD in aĀ reducedĀ state. ElectronsĀ flow from TMPD to cytochrome c. | ||
For preparation and titration please visit [[Ascorbate]] | For preparation and titration please visit [[Ascorbate]] | ||
Revision as of 13:17, 26 March 2014
- 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 methods:
Respirometry
MitoPedia topics:
Substrate and metabolite
Application in HRR
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.
- 1) Weigh 47.4 mg TMPD
- 2) Dissolve in 1 ml H2O
- 3) Divide into 0.2 ml portions
- 4) Protect from light!
- 5) Store frozen at -20 Ā°C
Caution: Light sensitive (store solution in a dark glass vial). Take care to minimize light and temperature exposure during use!
It is important to add Ascorbateshortly before TMPD to avoid uncontrollable autoxidation. Ascorbate maintains TMPD in a reduced state. Electrons flow from TMPD to cytochrome c.
For preparation and titration please visit Ascorbate
Oxygraph-2k Manual Titrations: MiPNet09.12
- Titration volume: 5 Āµl using a 25 Āµl syringe
- Final conc. in 2 ml O2k-chamber: 0.5 mM
