Difference between revisions of "TMPD"

Revision as of 11:41, 22 October 2020

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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; FW = 237.2.

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. 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:

Weigh 47.4 mg TMPD
Dissolve in 1 mL H₂O
Divide into 0.2 mL portions
Protect from light!
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:

Weigh 59,25 mg TMPD
Dissolve in 1 mL H₂O
Divide into 0.2 mL portions
Protect from light!
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

@@ Line 9: / Line 9: @@
 ::: '''TMPD''' (C<sub>10</sub>H<sub>16</sub>N<sub>2</sub>*2HCl): Sigma T 3134, 5 g, store at RT; FW = 237.2.
 :::: <span  style="color:#8B008B"> '''Caution:''' Light sensitive (store  solution in a dark glass vial). Take care to minimize light and  temperature exposure during use!</span>
-:::''' Complex IV assay '''
+::::''' 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.
-::::* 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.
-::::''' Preparation of 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]]
+::::''' Preparation of 200 mM stock solution''' (dissolved in H<sub>2</sub>O) for '''2-mL O2k-chamber''':
 ::::# Weigh 47.4 mg TMPD
 ::::# Dissolve in 1 mL H<sub>2</sub>O
@@ Line 21: / Line 23: @@
 :::» '''O2k manual titrations '''  [[MiPNet09.12 O2k-Titrations]]
-::::* Titration volume: 5 µL using a 25 µL syringe.
+::::* Titration volume ('''2-mL O2k-chamber'''): 5 µL using a 25 µL Hamilton syringe.
-::::* Final conc. in 2 mL O2k-chamber: 0.5 mM.
+::::* Final concentration: 0.5 mM.
+::::''' 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 1 mL H<sub>2</sub>O
+::::# Divide into 0.2 mL portions
+::::# Protect from light!
+::::# Store frozen at -20 °C
+:::» '''O2k manual titrations '''  [[MiPNet09.12 O2k-Titrations]]
-:::: 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''.
+::::* Titration volume ('''0.5-mL O2k-chamber'''): 1 µL using a 10 µL Hamilton syringe.
+::::* Final concentration: 0.5 mM.
-:::: For preparation and titration please visit [[Ascorbate]]
 {{MitoPedia topics
 |mitopedia topic=Substrate and metabolite
 }}