Difference between revisions of "TMPD"
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=== Complex IV assay === | === Complex IV assay === | ||
* [[Complex IV]] | * For theĀ [[Complex_IV#HRR_protocol:_Complex_IV_assay|Complex IV assay]] TMPD isĀ required at 0.5 mM final concentration added from a 200 mM stock. | ||
=== Preparation of 200 mM stock === | === Preparation of 200 mM stock === | ||
TMPD (C<sub>10</sub>H<sub>16</sub>N<sub>2</sub>*2HCl): Sigma T 3134, 5 g, store at RT; FW = 237.2. | TMPD Ā (C<sub>10</sub>H<sub>16</sub>N<sub>2</sub>*2HCl): Ā Sigma T 3134, 5 g, store at RT; FW = 237.2. | ||
Ā Ā | Ā Ā | ||
::1) Weigh 47.4 mg TMPD | ::1) Weigh 47.4 mg TMPD | ||
::2) Dissolve in 1 ml H<sub>2</sub>O Ā | ::2) Dissolve in 1 ml H<sub>2</sub>O Ā | ||
::3) | ::3) Divide into 0.2 ml portions | ||
::4) | ::4) Protect from light! | ||
::5) Store frozen at -20 Ā°C | ::5) Store frozen at -20 Ā°C | ||
<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. | |||
For preparation and titration please visit [[Ascorbate]] | |||
Revision as of 10:23, 20 March 2014
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 Ascorbate 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