Difference between revisions of "TMPD"
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{{MitoPedia | {{MitoPedia | ||
|abbr=Tm | |abbr=Tm | ||
|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 [[Complex IV|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. | |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 [[Complex IV|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. | ||
|info=[[MiPNet06.06]], [[MiPNet09.12]] | |info=[[MiPNet06.06]], [[MiPNet09.12]] | ||
}} | }} | ||
{{MitoPedia topics | {{MitoPedia topics | ||
|mitopedia topic=Substrate and metabolite | |mitopedia topic=Substrate and metabolite | ||
}} | }} | ||
__TOC__ | __TOC__ | ||
== Application in [[ | == Application in [[HRFR]] == | ||
=== Complex IV assay === | === Complex IV assay === | ||
* For the [[Complex_IV#HRR_protocol:_Complex_IV_assay|Complex IV assay]] TMPD is required at 0.5 mM final concentration added from a 200 mM stock. | ::::* For the [[Complex_IV#HRR_protocol:_Complex_IV_assay|Complex IV assay]] TMPD is required at 0.5 mM final concentration added from a 200 mM stock. | ||
=== Preparation of 200 mM stock === | === Preparation of 200 mM stock === | ||
TMPD | ::: 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. | ||
:: | ::::# Weigh 47.4 mg TMPD | ||
:: | ::::# Dissolve in 1 ml H<sub>2</sub>O | ||
:: | ::::# Divide into 0.2 ml portions | ||
:: | ::::# Protect from light! | ||
:: | ::::# Store frozen at -20 °C | ||
<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> | ||
It is | :::: It is important to add [[Ascorbate]] 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]] | :::: For preparation and titration please visit [[Ascorbate]] | ||
'''Oxygraph-2k Manual Titrations:''' [[MiPNet09.12]] | ::: '''Oxygraph-2k Manual Titrations:''' [[MiPNet09.12]] | ||
::* 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. |
Revision as of 09:41, 6 August 2017
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!
- It is important to add Ascorbate 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
- 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.