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 (final conc. 280 units/ml) directly to the MiR05 stock solution

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

Preparation of catalase stock solution

Addition of catalase to MiR05 stock solution of 1000 ml final volume:

Compound	Final conc.	Addition to 1 litre of MiR05	Company, product code and storage
Catalase lypophilized powder	280 units/ml	100 mg ('units/mg solid' is used for calculations)	Sigma C 9322, store at -20 °C
2800 units/mg solid; 3500 units/mg protein

Addition of catalase stock solution into the 2 ml oxygraph chamber:

Compound	Final conc.	Stock solution	Addition to 2 ml final volume (oxygraph chamber)
Catalase lypophilized powder	280 units/ml	40 mg dissolved in 1 ml MiR05 = 112000 units/ml	5 µl

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

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