TMPD: Difference between revisions
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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|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]] | ||
}} | }} | ||
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::::''' Complex IV assay ''':Β 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. | ::::''' Complex IV assay ''':Β 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. | ||
:::: 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 | :::: 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, and electrons flow from TMPD to cytochrome ''c''. For preparation and titration please visit [[Ascorbate]]. | ||
Revision as of 12:50, 5 March 2021
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; M = 237.2 gΒ·mol-1.
- 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; M = 237.2 gΒ·mol-1.
- Preparation of 200 mM stock solution (dissolved in H2O) for 2-mL O2k-chamber:
- 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 solution (dissolved in H2O) for 2-mL O2k-chamber:
- Β» O2k manual titrations MiPNet09.12 O2k-Titrations
- Titration volume (2-mL O2k-chamber): 5 Β΅L using a 25 Β΅L Hamilton syringe.
- Final concentration: 0.5 mM.
- Preparation of 250 mM stock solution (dissolved in H2O) for 0.5-mL O2k-chamber:
- Weigh 59,25 mg TMPD
- Dissolve in 1 mL H2O
- Divide into 0.2 mL portions
- Protect from light!
- Store frozen at -20 Β°C
- Β» O2k manual titrations MiPNet09.12 O2k-Titrations
- Titration volume (0.5-mL O2k-chamber): 1 Β΅L using a 10 Β΅L Hamilton syringe.
- Final concentration: 0.5 mM.
MitoPedia topics: Substrate and metabolite