TMPD: Difference between revisions
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== Application in [[HRR]]Β == | == Application in [[HRR]]Β == | ||
Β | ::: '''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. | ||
= | :::: <spanΒ style="color:#8B008B"> '''Caution:''' Light sensitive (storeΒ solution in a dark glass vial). Take care to minimize light andΒ temperature exposure during use!</span> | ||
:::''' Complex IV assay ''' | |||
::::* For theΒ [[Complex_IV#HRR_protocol:_Complex_IV_assay|Complex IV assay]] TMPD isΒ required at 0.5 mM final concentration added from a 200 mM stock. | ::::* For theΒ [[Complex_IV#HRR_protocol:_Complex_IV_assay|Complex IV assay]] TMPD isΒ required at 0.5 mM final concentration added from a 200 mM stock. | ||
::::''' Preparation of 200 mM stock ''' | |||
::::# Weigh 47.4 mg TMPD | ::::# Weigh 47.4 mg TMPD | ||
::::# Dissolve in 1 mL H<sub>2</sub>O Β | ::::# Dissolve in 1 mL H<sub>2</sub>O Β | ||
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::::# Store frozen at -20 Β°C | ::::# Store frozen at -20 Β°C | ||
::: | :::Β» '''O2k manual titrations '''Β [[MiPNet09.12 O2k-Titrations]] | ||
Β | |||
Β | |||
Β | |||
::::* Titration volume: 5 Β΅L using a 25 Β΅L syringe. | ::::* Titration volume: 5 Β΅L using a 25 Β΅L syringe. |
Revision as of 09:49, 11 September 2020
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; FW = 237.2.
- 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; FW = 237.2.
- Preparation of 200 mM stock
- Weigh 47.4 mg TMPD
- Dissolve in 1 mL H2O
- Divide into 0.2 mL portions
- Protect from light!
- Store frozen at -20 Β°C
- Preparation of 200 mM stock
- Β» O2k manual titrations MiPNet09.12 O2k-Titrations
- Titration volume: 5 Β΅L using a 25 Β΅L 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
MitoPedia topics:
Substrate and metabolite