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