From Bioblast
Revision as of 09:25, 15 September 2021 by Spitzer Gudrun (talk | contribs) (added 'h' to the H-statement numbers)
- 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
- 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!
- 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 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 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.
- 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 µL H2O
- 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.
- Final concentration: 0.5 mM.
MitoPedia topics: Substrate and metabolite
