MiR06

high-resolution terminology - matching measurements at high-resolution

MiR06

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 (MiR06 = MiR05 + Catalase). Total volume = 1 litre.

Oxygen solubility factor in MiR06 (MiR06) at 30 °C = 0.92

H₂O₂: Small volumes (µl) of H₂O₂ are injected in to the O2k-chamber filled with MiR06, to increase oxygen levels. A typical [[#Preparation of H₂O₂ 200 mM 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 °C

4) add 120 ml of K-lactobionate stock solution

5a) adjust the pH to 7.1 with 5 N KOH at 30 °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 °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 °C
K-lactobionate	60 mM	358.3	120 ml of 0.5 M K-lactobionate stock	Aldrich P 153516, 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 °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 µl 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 µmol/min; assay used by Sigma Aldrich: ' One unit will decompose 1.0 μmole of H₂O₂ per min at pH 7.0 at 25 °C, while the H₂O₂ concentration falls from 10.3 to 9.2 mM, measured by the rate of decrease of AH₂₄₀. '

Preparation of H₂O₂ 200 mM stock solution

H₂O₂: Hydrogen peroxide solution, 50 wt. % in H2O, stabilized, Sigma 516813, store in the fridge

1) pipette 114 µl of 17.6 M H₂O₂ into 10 ml plastic vial

2) add H₂O_{to a total volume of 10 ml}

3) wrap the 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.