Difference between revisions of "Cyanide"
From Bioblast
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|info=[[MiPNet09.12]] | |info=[[MiPNet09.12]] | ||
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== Application in [[HRR]] == | == Application in [[HRR]] == | ||
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::::* [[alternative oxidase]] | ::::* [[alternative oxidase]] | ||
::: ''' | :::: '''Kcn'''(KCN): Fluka 60178, 100 g, store at R.T.; FW = 65.12; '''Trizma base''': Sigma T6066, 100 g, store at R.T.; FW = 121.14 | ||
:::: | |||
:::: | :::: <span style="color:#8B008B"> '''Caution:''' KCN is photosensitive and hygroscopic; KCN is volatile and very toxic! It is recommended to prepare fresh KCN solution although it can be safely stored at -20°C. To avoid KCN evaporation, it is recommended to use alkaline buffers (such as 45 mM Trizma base, pH >10).</span> | ||
:::: '''Preparation of 45 mM Trizma base:''' | |||
::::# Weigh 545 mg of Trizma base | |||
::::# Dissolve it in 100 mL of H<sub>2</sub>0. | |||
::::''' Preparation of 20 mM KCN stock solution ''' (in 45 mM Trizma base) for '''2-mL O2k-chamber''': | |||
::::# Weigh 5.2 mg of KCN and dissolve in 4 mL of 45 mM Trizma base. | |||
::::# Divide into 500 µL aliquots (use dark glass vials). | |||
::::# Store frozen at -20 °C. | |||
:::: ''' | :::» '''O2k manual titrations''' [[MiPNet09.12 O2k-Titrations]] | ||
::::* Titration volume ('''2-mL O2k-chamber'''): 100 µL using a 100 µL Hamilton syringe. | |||
::::* Final concentration: 1 mM. | |||
::::# Weigh | :::: '''Preparation of 250 mM KCN stock solution''' (in 45 mM Trizma base) for '''0.5-mL O2k-chamber''': | ||
::::# | ::::# Weigh 16,28 mg of KCN and dissolve in 1 mL of 45 mM Trizma base. | ||
:::: | ::::# Divide into 200 µL aliquots (use dark glass vials). | ||
::::# Store frozen at -20 °C. | |||
:::» '''O2k manual titrations''' [[MiPNet09.12 O2k-Titrations]] | |||
::::* Titration volume ('''0.5-mL O2k-chamber'''): 2 µL using a 10 µL Hamilton syringe. | |||
::::* Final concentration: 1 mM. | |||
{{MitoPedia O2k and high-resolution respirometry|type=Inhibitor ETS | |||
}} | |||
{{MitoPedia topics | |||
|mitopedia topic=Inhibitor | |||
}} |
Revision as of 12:08, 22 January 2021
Description
Cyanide (usually added as KCN) is a competitive inhibitor of cytochcrome c oxidase (CIV). Inhibition is reversed by pyruvate and high oxygen levels.
Abbreviation: KCN
Reference: MiPNet09.12
Application in HRR
Inhibitor titrations, CIV-assay, alternative oxidase and ROX
- Cyanide and determination of residual oxygen consumption (ROX).
- Complex IV assay
- alternative oxidase
- Kcn(KCN): Fluka 60178, 100 g, store at R.T.; FW = 65.12; Trizma base: Sigma T6066, 100 g, store at R.T.; FW = 121.14
- Caution: KCN is photosensitive and hygroscopic; KCN is volatile and very toxic! It is recommended to prepare fresh KCN solution although it can be safely stored at -20°C. To avoid KCN evaporation, it is recommended to use alkaline buffers (such as 45 mM Trizma base, pH >10).
- Preparation of 45 mM Trizma base:
- Weigh 545 mg of Trizma base
- Dissolve it in 100 mL of H20.
- Preparation of 45 mM Trizma base:
- Preparation of 20 mM KCN stock solution (in 45 mM Trizma base) for 2-mL O2k-chamber:
- Weigh 5.2 mg of KCN and dissolve in 4 mL of 45 mM Trizma base.
- Divide into 500 µL aliquots (use dark glass vials).
- Store frozen at -20 °C.
- Preparation of 20 mM KCN stock solution (in 45 mM Trizma base) for 2-mL O2k-chamber:
- » O2k manual titrations MiPNet09.12 O2k-Titrations
- Titration volume (2-mL O2k-chamber): 100 µL using a 100 µL Hamilton syringe.
- Final concentration: 1 mM.
- » O2k manual titrations MiPNet09.12 O2k-Titrations
- Preparation of 250 mM KCN stock solution (in 45 mM Trizma base) for 0.5-mL O2k-chamber:
- Weigh 16,28 mg of KCN and dissolve in 1 mL of 45 mM Trizma base.
- Divide into 200 µL aliquots (use dark glass vials).
- Store frozen at -20 °C.
- Preparation of 250 mM KCN stock solution (in 45 mM Trizma base) for 0.5-mL O2k-chamber:
- » O2k manual titrations MiPNet09.12 O2k-Titrations
- Titration volume (0.5-mL O2k-chamber): 2 µL using a 10 µL Hamilton syringe.
- Final concentration: 1 mM.
- » O2k manual titrations MiPNet09.12 O2k-Titrations
MitoPedia topics:
Inhibitor