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.

Caution: Chemicals stored in the fridge or freezer should be adjusted to room temperature before opening.

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

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. '