TMPD: Difference between revisions
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== Application in [[HRR]] == | == Application in [[HRR]] == | ||
::: '''TMPD''' (C<sub>10</sub>H<sub>16</sub>N<sub>2</sub>*2HCl): Sigma T 3134, 5 g, store at RT; M = 237.2 g·mol<sup>-1</sup>. | |||
:::: <span style="color:#8B008B"> '''Caution:''' Sensitive to | {{Chemical_description | ||
::::''' Complex IV assay ''': For the [[Complex_IV#HRR_protocol:_Complex_IV_assay|Complex IV assay]] TMPD is | |abbr=Tm | ||
|trivial name=TMPD | |||
|complete name=''N,N,N′,N′''-Tetramethyl-''p''-phenylenediamine dihydrochloride | |||
|chem formula=C<sub>10</sub>H<sub>16</sub>N<sub>2</sub>*2HCl | |||
|molar mass=237.2 | |||
|vendor=Sigma-Aldrich | |||
|product number=T3134 | |||
|store at=RT | |||
|sensitivity= | |||
|cas=637-01-4 | |||
|h statements=H315, H319 | |||
|h info=causes skin irritation, causes serious eye irritation | |||
}}<!--::: '''TMPD''' (C<sub>10</sub>H<sub>16</sub>N<sub>2</sub>*2HCl): Sigma T 3134, 5 g, store at RT; M =237.2 g·mol<sup>-1</sup>.--> | |||
:::: <span style="color:#8B008B"> '''Caution:''' Sensitive to light and oxidation (store solution in a dark vials). Take care to minimize light, oxygen and temperature exposure during use!</span> | |||
::::''' Complex IV assay''': For the [[Complex_IV#HRR_protocol:_Complex_IV_assay|Complex IV assay]] TMPD is used at an experimental concentration of 0.5 mM in the O2k-chamber. The abbreviation TMPD is shortened further to the symbol Tm used in DatLab events and SUIT protocol diagrams. | |||
:::: 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]]. | :::: 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]]. | ||
:::: 2021-03: The preparation instructions were updated to take the volume of the solute ( | :::* 2024-05-03: Ascorbate should be added to the TMPD stock solution to maintain TMPD in a reduced state and protect it from oxidation. This was the procedure until 2014, when addition of ascorbate to the TMPD stock was discontinued for unknown reasons, as noticed in May 2024, when instructions were changed again to prepare and use the TMPD stock solution containing ascorbate. | ||
:::* 2021-03-9: The preparation instructions were updated to take into account the volume of the solute (TMPD; see: [[Volume of the solute]]). | |||
2024-05-03 to present | |||
::::''' Preparation of 200 mM stock solution''' (1 mL, dissolved in H<sub>2</sub>O) for '''2-mL O2k-chamber''': | ::::''' Preparation of 200 mM stock solution''' (1 mL, dissolved in H<sub>2</sub>O, with ascrobate) for the '''2-mL O2k-chamber''': | ||
::::# Weigh 47.4 mg TMPD | ::::# Weigh 47.4 mg TMPD | ||
::::# Dissolve in 970 | ::::# Dissolve in 970 mL H<sub>2</sub>O | ||
::::# Divide into 0.2 mL portions | ::::# Add 0.8 M [[ascorbate]] to a final concentration of 10 mM | ||
::::# Divide into 0.2 mL portions | |||
::::# Store frozen at -20 °C | ::::# Store frozen at -20 °C | ||
Line 25: | Line 45: | ||
::::* Titration volume ('''2-mL O2k-chamber'''): 5 µL using a 25 µL Hamilton syringe. | ::::* Titration volume ('''2-mL O2k-chamber'''): 5 µL using a 25 µL Hamilton syringe. | ||
::::* | ::::* Experimental concentration: 0.5 mM. | ||
::::''' Preparation of 250 mM stock solution''' (dissolved in H<sub>2</sub>O) for '''0.5-mL O2k-chamber''': | ::::''' Preparation of 250 mM stock solution''' (dissolved in H<sub>2</sub>O) for '''0.5-mL O2k-chamber''': | ||
::::# Weigh 59 | ::::# Weigh 59.25 mg TMPD | ||
::::# Dissolve in 962 µL H<sub>2</sub>O | ::::# Dissolve in 962 µL H<sub>2</sub>O | ||
::::# Add 0.8 M [[ascorbate]] to a final concentration of 10 mM | |||
::::# Divide into 0.2 mL portions in dark vials | ::::# Divide into 0.2 mL portions in dark vials | ||
::::# Store frozen at -20 °C | ::::# Store frozen at -20 °C | ||
Line 38: | Line 58: | ||
::::* Titration volume ('''0.5-mL O2k-chamber'''): 1 µL using a 10 µL Hamilton syringe. | ::::* Titration volume ('''0.5-mL O2k-chamber'''): 1 µL using a 10 µL Hamilton syringe. | ||
::::* | ::::* Experimental concentration: 0.5 mM. | ||
2021-03-09 to 2024-05-03 | |||
::::''' Preparation of 200 mM stock solution''' (1 mL, dissolved in H<sub>2</sub>O) for the '''2-mL O2k-chamber''': | |||
::::# Weigh 47.4 mg TMPD | |||
::::# Dissolve in 970 µL H<sub>2</sub>O | |||
::::# Divide into 0.2 mL portions in dark vials | |||
::::# Store frozen at -20 °C | |||
::::''' Preparation of 250 mM stock solution''' (dissolved in H<sub>2</sub>O) for '''0.5-mL O2k-chamber''': | |||
::::# Weigh 59.25 mg TMPD | |||
::::# Dissolve in 962 µL H<sub>2</sub>O | |||
::::# Divide into 0.2 mL portions in dark vials | |||
::::# Store frozen at -20 °C | |||
2014-03-20 to 2021-03-09 | |||
::::''' Preparation of 200 mM stock solution''' (1 mL, dissolved in H<sub>2</sub>O) for the '''2-mL O2k-chamber''': | |||
::::# Weigh 47.4 mg TMPD | |||
::::# Dissolve in 1 mL H<sub>2</sub>O | |||
::::# Divide into 0.2 mL portions in dark vials | |||
::::# Store frozen at -20 °C | |||
up to 2014-03-20 | |||
::::''' Preparation of 200 mM stock solution''' (1 mL, dissolved in H<sub>2</sub>O) for the '''2-mL O2k-chamber''': | |||
::::# Weigh 47.4 mg TMPD | |||
::::# Dissolve in 1 mL H<sub>2</sub>O | |||
::::# Add 0.8 M [[ascorbate]] to a final concentration of 10 mM | |||
::::# Divide into 0.2 mL portions in dark vials | |||
::::# Store frozen at -20 °C | |||
{{MitoPedia topics | {{MitoPedia topics | ||
|mitopedia topic=Substrate and metabolite | |mitopedia topic=Substrate and metabolite | ||
}} | }} |
Latest revision as of 10:08, 3 May 2024
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
- Tm: TMPD (N,N,N′,N′-Tetramethyl-p-phenylenediamine dihydrochloride; C10H16N2*2HCl), Sigma-Aldrich: T3134, store at RT, CAS: 637-01-4, M = 237.2 g·mol-1
- Hazard statements: H315, H319; causes skin irritation, causes serious eye irritation
- Tm: TMPD (N,N,N′,N′-Tetramethyl-p-phenylenediamine dihydrochloride; C10H16N2*2HCl), Sigma-Aldrich: T3134, store at RT, CAS: 637-01-4, M = 237.2 g·mol-1
- Caution: Sensitive to light and oxidation (store solution in a dark vials). Take care to minimize light, oxygen and temperature exposure during use!
- Complex IV assay: For the Complex IV assay TMPD is used at an experimental concentration of 0.5 mM in the O2k-chamber. The abbreviation TMPD is shortened further to the symbol Tm used in DatLab events and SUIT protocol diagrams.
- 2024-05-03: Ascorbate should be added to the TMPD stock solution to maintain TMPD in a reduced state and protect it from oxidation. This was the procedure until 2014, when addition of ascorbate to the TMPD stock was discontinued for unknown reasons, as noticed in May 2024, when instructions were changed again to prepare and use the TMPD stock solution containing ascorbate.
- 2021-03-9: The preparation instructions were updated to take into account the volume of the solute (TMPD; see: Volume of the solute).
2024-05-03 to present
- Preparation of 200 mM stock solution (1 mL, dissolved in H2O, with ascrobate) for the 2-mL O2k-chamber:
- Weigh 47.4 mg TMPD
- Dissolve in 970 mL H2O
- Add 0.8 M ascorbate to a final concentration of 10 mM
- Divide into 0.2 mL portions
- Store frozen at -20 °C
- Preparation of 200 mM stock solution (1 mL, dissolved in H2O, with ascrobate) for the 2-mL O2k-chamber:
- » O2k manual titrations MiPNet09.12 O2k-Titrations
- Titration volume (2-mL O2k-chamber): 5 µL using a 25 µL Hamilton syringe.
- Experimental 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 962 µL H2O
- Add 0.8 M ascorbate to a final concentration of 10 mM
- Divide into 0.2 mL portions in dark vials
- 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.
- Experimental concentration: 0.5 mM.
2021-03-09 to 2024-05-03
- Preparation of 200 mM stock solution (1 mL, dissolved in H2O) for the 2-mL O2k-chamber:
- Weigh 47.4 mg TMPD
- Dissolve in 970 µL H2O
- Divide into 0.2 mL portions in dark vials
- Store frozen at -20 °C
- Preparation of 200 mM stock solution (1 mL, dissolved in H2O) for the 2-mL O2k-chamber:
- Preparation of 250 mM stock solution (dissolved in H2O) for 0.5-mL O2k-chamber:
- Weigh 59.25 mg TMPD
- Dissolve in 962 µL H2O
- Divide into 0.2 mL portions in dark vials
- Store frozen at -20 °C
2014-03-20 to 2021-03-09
- Preparation of 200 mM stock solution (1 mL, dissolved in H2O) for the 2-mL O2k-chamber:
- Weigh 47.4 mg TMPD
- Dissolve in 1 mL H2O
- Divide into 0.2 mL portions in dark vials
- Store frozen at -20 °C
- Preparation of 200 mM stock solution (1 mL, dissolved in H2O) for the 2-mL O2k-chamber:
up to 2014-03-20
- Preparation of 200 mM stock solution (1 mL, dissolved in H2O) for the 2-mL O2k-chamber:
- Weigh 47.4 mg TMPD
- Dissolve in 1 mL H2O
- Add 0.8 M ascorbate to a final concentration of 10 mM
- Divide into 0.2 mL portions in dark vials
- Store frozen at -20 °C
- Preparation of 200 mM stock solution (1 mL, dissolved in H2O) for the 2-mL O2k-chamber:
MitoPedia topics:
Substrate and metabolite