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.

Chemicals for the preparation of MiR05 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 the powder by stirring on a magnetic stirrer at ~30 °C

4) add 120 ml of K-lactobionate stock solution

5) adjust the pH to 7.1 with 5 N KOH at 30 °C

6) divide in to 20 ml portions and store at -20 °C in plastic vials


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

Preparation of 5 N KOH stock solution

KOH (Potassium hydroxide, ≥85% KOH basis, pellets); Aldrich P 1767, 1000 g, store at R.T.: FW = 56.11