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Difference between revisions of "TMPD"

From Bioblast
(replaced FW by M and added units g*mol-1)
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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 ''':  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.
::::''' 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.


:::: 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]]
:::: 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 11:50, 5 March 2021


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

Application in HRR

TMPD (C10H16N2*2HCl): Sigma T 3134, 5 g, store at RT; M = 237.2 g·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.
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.


Preparation of 200 mM stock solution (dissolved in H2O) for 2-mL O2k-chamber:
  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 °C
» O2k manual titrations MiPNet09.12 O2k-Titrations
  • Titration volume (2-mL O2k-chamber): 5 µL using a 25 µL Hamilton syringe.
  • Final concentration: 0.5 mM.


Preparation of 250 mM stock solution (dissolved in H2O) for 0.5-mL O2k-chamber:
  1. Weigh 59,25 mg TMPD
  2. Dissolve in 1 mL H2O
  3. Divide into 0.2 mL portions
  4. Protect from light!
  5. Store frozen at -20 °C
» O2k manual titrations MiPNet09.12 O2k-Titrations
  • Titration volume (0.5-mL O2k-chamber): 1 µL using a 10 µL Hamilton syringe.
  • Final concentration: 0.5 mM.


MitoPedia topics: Substrate and metabolite