Difference between revisions of "Ascorbate"
From Bioblast
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{{MitoPedia | {{MitoPedia | ||
|abbr=As | |abbr=As | ||
|description=In respiratory assays for cytochrome c oxidase ( | |description=In respiratory assays for cytochrome ''c'' oxidase activity ([[Complex IV|Complex IV, CIV]]), '''ascorbate''' is added as regenerating system for maintaining [[TMPD]] in a reduced state. As has to be titrated into the respiration medium prior to the addition of TMPD, otherwise the reaction velocity of [[autoxidation]] is permanently elevated. | ||
|info=[[MiPNet06.06]] | |info=[[MiPNet06.06]] | ||
}} | }} | ||
{{MitoPedia methods}} | |||
{{MitoPedia topics | {{MitoPedia topics | ||
|mitopedia topic=Substrate and metabolite | |mitopedia topic=Substrate and metabolite | ||
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== Application in [[HRR]] == | == Application in [[HRR]] == | ||
'''As: Ascorbate sodium salt''' (Succinate disodium salt, hexahydrate, C<sub>6</sub>H<sub>7</sub>0<sub>6</sub>Na); Sigma A 4034, 100 g, store at | '''As: Ascorbate sodium salt''' (Succinate disodium salt, hexahydrate, C<sub>6</sub>H<sub>7</sub>0<sub>6</sub>Na); Sigma A 4034, 100 g, store at RT; FW = 198.1. | ||
<span style="color:#8B008B"> '''Caution:''' Light sensitive (store solution in a dark glass vial).</span> | <span style="color:#8B008B"> '''Caution:''' Light sensitive (store solution in a dark glass vial).</span> | ||
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'''Preparation of 800 mM stock solution''' (dissolved in H<sub>2</sub>O): | '''Preparation of 800 mM stock solution''' (dissolved in H<sub>2</sub>O): | ||
::1) weigh 1.584 g of | ::1) weigh 1.584 g of ascorbate sodium salt and dissolve in 10 ml H<sub>2</sub>O | ||
::2) To prevent | ::2) To prevent autoxidation, adjust pH to ca 6 with ascorbic acid (a 137.6 mg.ml<sup>-1</sup> solution of pH 2). | ||
::3) Divide into 0.2 ml portions. | ::3) Divide into 0.2 ml portions. | ||
::4) Store frozen at -20 °C. | ::4) Store frozen at -20 °C. | ||
'''Oxygraph-2k | '''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: 2 mM | ::* Final conc. in 2 ml O2k-chamber: 2 mM | ||
Revision as of 10:24, 25 May 2012
- high-resolution terminology - matching measurements at high-resolution
Ascorbate
Description
In respiratory assays for cytochrome c oxidase activity (Complex IV, CIV), ascorbate is added as regenerating system for maintaining TMPD in a reduced state. As has to be titrated into the respiration medium prior to the addition of TMPD, otherwise the reaction velocity of autoxidation is permanently elevated.
Abbreviation: As
Reference: MiPNet06.06
MitoPedia topics: Substrate and metabolite
Application in HRR
As: Ascorbate sodium salt (Succinate disodium salt, hexahydrate, C6H706Na); Sigma A 4034, 100 g, store at RT; FW = 198.1.
Caution: Light sensitive (store solution in a dark glass vial).
Preparation of 800 mM stock solution (dissolved in H2O):
- 1) weigh 1.584 g of ascorbate sodium salt and dissolve in 10 ml H2O
- 2) To prevent autoxidation, adjust pH to ca 6 with ascorbic acid (a 137.6 mg.ml-1 solution of pH 2).
- 3) Divide into 0.2 ml portions.
- 4) Store frozen at -20 °C.
Oxygraph-2k manual titrations: MiPNet09.12
- Titration volume: 5 µl using a 25 µl syringe
- Final conc. in 2 ml O2k-chamber: 2 mM
