Ascorbate: Difference between revisions
From Bioblast
No edit summary |
No edit summary |
||
Line 12: | Line 12: | ||
:::: <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> | ||
<!-- | |||
:::: '''Preparation of 800 mM stock solution''' (dissolved in H<sub>2</sub>O): | |||
::::# Weigh 1.584 g of ascorbate sodium salt and dissolve in 10 mL H<sub>2</sub>O | ::::# Weigh 1.584 g of ascorbate sodium salt and dissolve in 10 mL H<sub>2</sub>O | ||
::::# To prevent autoxidation, adjust pH to approx. 6 with ascorbic acid (a 137.6 mg.mL<sup>-1</sup> solution of pH 2). ย | ::::# To prevent autoxidation, adjust pH to approx. 6 with ascorbic acid (a 137.6 mg.mL<sup>-1</sup> solution of pH 2). ย | ||
::::# Divide into 0.2 mL portions. ย | ::::# Divide into 0.2 mL portions. ย | ||
::::# Store frozen at -20 ยฐC. ย | ::::# Store frozen at -20 ยฐC. ย | ||
:::ยป '''O2k manual titrations:''' [[MiPNet09.12]] | :::ยป '''O2k manual titrations:''' [[MiPNet09.12]] | ||
Line 24: | Line 24: | ||
::::* Titration volume: 5 ยตL using a 25 ยตL syringe. | ::::* Titration volume: 5 ยตL using a 25 ยตL syringe. | ||
::::* Final conc. in 2 mL O2k-chamber: 2 mM. | ::::* Final conc. in 2 mL O2k-chamber: 2 mM. | ||
--> | |||
:::: '''Preparation of 800 mM stock solution''' (dissolved in H<sub>2</sub>O) for '''2-mL O2k-chamber''': | |||
::::# Weigh 1.584 g of ascorbate sodium salt and dissolve in 10 mL H<sub>2</sub>O | |||
::::# To prevent autoxidation, adjust pH to approx. 6 with ascorbic acid (a 137.6 mg.mL<sup>-1</sup> solution of pH 2). | |||
::::# Divide into 0.2 mL portions. | |||
::::# Store frozen at -20 ยฐC. | |||
:::ยป'''O2k manual titrations:''' [[MiPNet09.12]] | |||
::::* Titration volume ('''2-mL O2k-chamber'''): 5 ยตL using a 25 ยตL Hamilton syringe. | |||
::::* Final concentration: 2 mM. | |||
:::: '''Preparation of 1000 mM stock solution''' (dissolved in H<sub>2</sub>O) for '''0.5-mL O2k-chamber''': | |||
::::# Weigh 1.980 g of ascorbate sodium salt and dissolve in 10 mL H<sub>2</sub>O | |||
::::# To prevent autoxidation, adjust pH to approx. 6 with ascorbic acid (a 137.6 mg.mL<sup>-1</sup> solution of pH 2). | |||
::::# Divide into 0.2 mL portions. | |||
::::# Store frozen at -20 ยฐC. | |||
:::ยป'''O2k manual titrations:''' [[MiPNet09.12]] | |||
::::* Titration volume ('''0.5-mL O2k-chamber'''): 1 ยตL using a 10 ยตL Hamilton syringe. | |||
::::* Final concentration: 2 mM. | |||
{{MitoPedia topics | {{MitoPedia topics | ||
|mitopedia topic=Substrate and metabolite | |mitopedia topic=Substrate and metabolite | ||
}} | }} |
Revision as of 11:25, 22 October 2020
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
Application in HRR
- As: Ascorbate sodium salt (Sodium L-ascorbate); Sigma A 7631, 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) for 2-mL O2k-chamber:
- Weigh 1.584 g of ascorbate sodium salt and dissolve in 10 mL H2O
- To prevent autoxidation, adjust pH to approx. 6 with ascorbic acid (a 137.6 mg.mL-1 solution of pH 2).
- Divide into 0.2 mL portions.
- Store frozen at -20 ยฐC.
- ยปO2k manual titrations: MiPNet09.12
- Titration volume (2-mL O2k-chamber): 5 ยตL using a 25 ยตL Hamilton syringe.
- Final concentration: 2 mM.
- Preparation of 1000 mM stock solution (dissolved in H2O) for 0.5-mL O2k-chamber:
- Weigh 1.980 g of ascorbate sodium salt and dissolve in 10 mL H2O
- To prevent autoxidation, adjust pH to approx. 6 with ascorbic acid (a 137.6 mg.mL-1 solution of pH 2).
- Divide into 0.2 mL portions.
- Store frozen at -20 ยฐC.
- ยปO2k manual titrations: MiPNet09.12
- Titration volume (0.5-mL O2k-chamber): 1 ยตL using a 10 ยตL Hamilton syringe.
- Final concentration: 2 mM.
MitoPedia topics:
Substrate and metabolite