MiR06: Difference between revisions
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'''MiR06: Mitochondrial Respiration Medium''' ([[#Preparation of MiR05 (MiR06) stock solution|MiR06]] = MiR05 + Catalase). Total volume = 1 litre. | '''MiR06: Mitochondrial Respiration Medium''' ([[#Preparation of MiR05 (MiR06) stock solution|MiR06]] = MiR05 + Catalase). Total volume = 1 litre. | ||
::Oxygen solubility factor in MiR06 (MiR06) at | ::Oxygen solubility factor in MiR06 (MiR06) at 30Β°C and 37Β°C = 0.92 | ||
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|align="right"|Sigma A 6003 fraction V, 25 g, store at 2-8 Β°C | |align="right"|Sigma A 6003 fraction V, 25 g, store at 2-8 Β°C | ||
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|align="right"| | |align="right"|Lactobionic acid | ||
|align="right"|60 mM | |align="right"|60 mM | ||
|align="right"|358.3 | |align="right"|358.3 | ||
|align="right"|120 ml of 0.5 M [[#Preparation of K-lactobionate stock solution|K-lactobionate stock]] | |align="right"|120 ml of 0.5 M [[#Preparation of K-lactobionate stock solution|K-lactobionate stock]] | ||
|align="right"|Aldrich | |align="right"|Aldrich 153516 or Sigma L2398, 100 g, store at R.T. | ||
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Revision as of 12:25, 24 November 2010
Description
Mitochondrial respiration medium 06, developed for oxygraph incubations of mitochondrial preparations = MiR05 plus catalase.
Abbreviation: MiR06
Reference: MiPNet14.13; Gnaiger 2000 MitoInTheCold
Labels:
HRR: Chemicals; Media"Chemicals; Media" is not in the list (Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, O2k-Spectrophotometer, O2k-Manual, O2k-Protocol, ...) of allowed values for the "Instrument and method" property.
Application in HRR
MiR06: Mitochondrial Respiration Medium (MiR06 = MiR05 + Catalase). Total volume = 1 litre.
- Oxygen solubility factor in MiR06 (MiR06) at 30Β°C and 37Β°C = 0.92
H2O2: Small volumes (Β΅l) of H2O2 are injected in to the O2k-chamber filled with MiR06, to increase oxygen levels. A typical [[#Preparation of 200 mM H2O2 stock solution|H2O2 stock concentration]] is 200 mM.
Preparation of MiR05 (MiR06) stock solution
- 1) weigh given amounts of the listed chemicals (except K-lactobionate) and transfer to a 1000 ml glass beaker
- 2) disrupt big lumps mechanically. It is recommended to do this before adding water, because during dissolution these lumps do not disintegrate easily.
- 3) add ~800 ml H2O and dissolve on a magnetic stirrer at ~30 Β°C
- 4) add 120 ml of K-lactobionate stock solution
- 5a) adjust the pH to 7.1 with 5 N KOH at 30 Β°C = MiR05
- 5b) to prepare MiR06 add catalase at a final concentration of 280 units*/ml) directly to the MiR05 stock solution (100 mg of 'Catalase, 2800 units/mg solid, Sigma C 9322')
- 6) divide into 20 ml portions and store at -20 Β°C in plastic vials
- MiR06 can also be prepared by adding catalase stock solution directly into the O2k-chamber filled with MiR05 at the start of the experiment.
Chemicals for MiR05
Caution: Chemicals stored in the fridge or freezer should be adjusted to room temperature before opening.
Compound | Final conc. | FW | Addition to 1 litre final volume | Company, product code and storage |
---|---|---|---|---|
EGTA | 0.5 mM | 380.4 | 0.190 g | Sigma E 4378, 25 g, store at R.T. |
MgCl2.6H20 | 3 mM | 203.3 | 0.610 g | Scharlau MA 0036, 250 g, store at R.T. |
Taurine | 20 mM | 125.1 | 2.502 g | Sigma T 0625, 25 g, store at R.T. |
KH2PO4 | 10 mM | 136.1 | 1.361 g | Merck 104873, 1000 g, store at R.T. |
HEPES | 20 mM | 238.3 | 4.77 g | Sigma H 7523, 250 g, store at R.T. |
D-Sucrose | 110 mM | 342.3 | 37.65 g | Roth, 4621.1, 1000 g, store at R.T. |
BSA essentially fatty acid free | 1 g/l | 1 g | Sigma A 6003 fraction V, 25 g, store at 2-8 Β°C | |
Lactobionic acid | 60 mM | 358.3 | 120 ml of 0.5 M K-lactobionate stock | Aldrich 153516 or Sigma L2398, 100 g, store at R.T. |
Preparation of K-lactobionate stock solution
- 1) weigh 35.83 g K-lactobionate directly into a 250 ml glass beaker
- 2) add 100 ml H2O and dissolve by stirring on magnetic stirrer
- 2) check pH (is approx. around 2.0) and neutralize with 5 N KOH
- 4) adjust final volume to 200 ml with H2O. It is best to use a 200 ml volumetric glass flask.
- 5) check pH again and adjust to 7 if necessary (5 N KOH)
Preparation of catalase stock solution
- Catalase lypophilized powder, 2800 units/mg solid; 3500 units/mg protein, Sigma C 9322, store at -20 Β°C
- Final conc.: 280 units/ml
- 1) Stock solution: dissolve 40 mg catalase powder in 1 ml MiR05 = 112000 units/ml
- 2) MiR06: add 5 Β΅l of the stock solution to the 2 ml oxygraph chamber filled with MiR05 to receive a final conc. of 280 units/ml
Unit definition: * Units of enzymatic activitiy (u) in Β΅mol/min; assay used by Sigma Aldrich: ' One unit will decompose 1.0 ΞΌmole of H2O2 per min at pH 7.0 at 25 Β°C, while the H2O2 concentration falls from 10.3 to 9.2 mM, measured by the rate of decrease of AH240. '
Preparation of 200 mM H2O2 stock solution
H2O2: Hydrogen peroxide solution, 50 wt. % in H2O, stabilized, Sigma 516813, store in the fridge
- 1) pipette 114 Β΅l of 17.6 M H2O2 into 10 ml plastic vial
- 2) add H2O to a total volume of 10 ml
- 3) wrap plastic vial in tin foil (solution is light sensitive) and store in the fridge
- 4) during experiments keep the solution on ice to prevent from bubble forming
For detailed information on chemicals see MiPNet14.13.