TMPD: Difference between revisions

Revision as of 09:41, 6 August 2017

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 HRFR

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!

It is important to add Ascorbate 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

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.

@@ Line 1: / Line 1: @@
 {{MitoPedia
 |abbr=Tm
-|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 [[Complex IV|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.
+|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 [[Complex IV|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.
 |info=[[MiPNet06.06]], [[MiPNet09.12]]
-|type=Substrate ETS
-}}
-{{MitoPedia concepts|type=Substrate ETS
-}}
-{{MitoPedia methods|type=Substrate ETS
-}}
-{{MitoPedia O2k and high-resolution respirometry|type=Substrate ETS
 }}
 {{MitoPedia topics
 |mitopedia topic=Substrate and metabolite
-|type=Substrate ETS
 }}
 __TOC__
-== Application in [[HRR]]  ==
+== Application in [[HRFR]]  ==
 === 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 ===
-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.
+::: 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.
-::1) Weigh 47.4 mg TMPD
+::::# Weigh 47.4 mg TMPD
-::2) Dissolve in 1 ml H<sub>2</sub>O
+::::# Dissolve in 1 ml H<sub>2</sub>O
-::3) Divide into 0.2 ml portions
+::::# Divide into 0.2 ml portions
-::4) Protect from light!
+::::# Protect from light!
-::5) Store frozen at -20 °C
+::::# Store frozen at -20 °C
-<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>
+:::: <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>
-It is  important to add  [[Ascorbate]] shortly before TMPD  to avoid  uncontrollable  autoxidation.  Ascorbate maintains TMPD in a  reduced  state. Electrons  flow from TMPD to cytochrome c.
+:::: It is important to add [[Ascorbate]] 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]]
-'''Oxygraph-2k Manual Titrations:'''  [[MiPNet09.12]]
+::: '''Oxygraph-2k Manual Titrations:'''  [[MiPNet09.12]]
-::* Titration volume: 5 µl using a 25 µl syringe
+::::* Titration volume: 5 µl using a 25 µl syringe.
-::* Final conc. in 2 ml O2k-chamber: 0.5 mM
+::::* Final conc. in 2 ml O2k-chamber: 0.5 mM.