TMPD: Difference between revisions
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}}<!--::: '''TMPD''' (C<sub>10</sub>H<sub>16</sub>N<sub>2</sub>*2HCl): Sigma T 3134, 5 g, store at RT; M =237.2ย gยทmol<sup>-1</sup>.--> | }}<!--::: '''TMPD''' (C<sub>10</sub>H<sub>16</sub>N<sub>2</sub>*2HCl): Sigma T 3134, 5 g, store at RT; M =237.2ย gยทmol<sup>-1</sup>.--> |
Revision as of 09:25, 15 September 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
- Tm: TMPD (N,N,Nโฒ,Nโฒ-Tetramethyl-p-phenylenediamine dihydrochloride; C10H16N2*2HCl), Sigma-Aldrich: T3134, store at RT, CAS: 637-01-4, M = 237.2 gยทmol-1
- Hazard statements: H315, H319; causes skin irritation, causes serious eye irritation
- Tm: TMPD (N,N,Nโฒ,Nโฒ-Tetramethyl-p-phenylenediamine dihydrochloride; C10H16N2*2HCl), Sigma-Aldrich: T3134, store at RT, CAS: 637-01-4, M = 237.2 gยทmol-1
- Caution: Sensitive to light and oxidation (store solution in a dark vials). Take care to minimize light, oxygen and temperature exposure during use!
- 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.
- Complex IV assay: For the Complex IV assay TMPD is required at 0.5 mM final concentration added from a 200 mM stock.
- 2021-03: The preparation instructions were updated to take the volume of the solute (TMPD) into account (see: Volume of the solute).
- Preparation of 200 mM stock solution (1 mL, dissolved in H2O) for 2-mL O2k-chamber:
- Weigh 47.4 mg TMPD
- Dissolve in 970 ยตL H2O
- Divide into 0.2 mL portions in dark vials
- Store frozen at -20 ยฐC
- Preparation of 200 mM stock solution (1 mL, dissolved in H2O) for 2-mL O2k-chamber:
- ยป O2k manual titrations MiPNet09.12 O2k-Titrations
- Titration volume (2-mL O2k-chamber): 5 ยตL using a 25 ยตL 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 962 ยตL H2O
- Divide into 0.2 mL portions in dark vials
- Store frozen at -20 ยฐC
- ยป O2k manual titrations MiPNet09.12 O2k-Titrations
- Titration volume (0.5-mL O2k-chamber): 1 ยตL using a 10 ยตL Hamilton syringe.
- Final concentration: 0.5 mM.
MitoPedia topics: Substrate and metabolite