TMPD: Difference between revisions
From Bioblast
No edit summary |
No edit summary |
||
| Line 8: | Line 8: | ||
}} | }} | ||
__TOC__ | __TOC__ | ||
== Application in [[ | == Application in [[HRR]] == | ||
=== Complex IV assay === | === Complex IV assay === | ||
| Line 17: | Line 17: | ||
::::# Weigh 47.4 mg TMPD | ::::# Weigh 47.4 mg TMPD | ||
::::# Dissolve in 1 | ::::# Dissolve in 1 mL H<sub>2</sub>O | ||
::::# Divide into 0.2 | ::::# Divide into 0.2 mL portions | ||
::::# Protect from light! | ::::# Protect from light! | ||
::::# Store frozen at -20 °C | ::::# Store frozen at -20 °C | ||
| Line 28: | Line 28: | ||
::: '''Oxygraph-2k Manual Titrations:''' [[MiPNet09.12]] | ::: '''Oxygraph-2k Manual Titrations:''' [[MiPNet09.12]] | ||
::::* Titration volume: 5 | ::::* Titration volume: 5 µL using a 25 µL syringe. | ||
::::* Final conc. in 2 | ::::* Final conc. in 2 mL O2k-chamber: 0.5 mM. | ||
:::: 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''. | :::: 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]] | :::: For preparation and titration please visit [[Ascorbate]] | ||
Revision as of 13:24, 17 March 2019
- 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
MitoPedia topics:
Substrate and metabolite
Application in HRR
Complex IV assay
- For the Complex IV assay TMPD is required at 0.5 mM final concentration added from a 200 mM stock.
Preparation of 200 mM stock
- TMPD (C10H16N2*2HCl): Sigma T 3134, 5 g, store at RT; FW = 237.2.
- Weigh 47.4 mg TMPD
- Dissolve in 1 mL H2O
- Divide into 0.2 mL portions
- Protect from light!
- Store frozen at -20 °C
- Caution: Light sensitive (store solution in a dark glass vial). Take care to minimize light and temperature exposure during use!
- Oxygraph-2k Manual Titrations: MiPNet09.12
- Titration volume: 5 µL using a 25 µL syringe.
- Final conc. in 2 mL O2k-chamber: 0.5 mM.
- 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
