Difference between revisions of "MiR06"
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=== Preparation of MiR07 ===
=== Preparation of MiR07 ===
::1) fresh 3 mg/mL creatine monohydrate (Fluka 27900) MiR06
::2) gently on magnetic stirrer
Revision as of 09:11, 30 November 2011
MitoPedia methods: Respirometry
MitoPedia topics: Media for respirometry
Application in HRR
- Oxygen solubility factor in MiR06 (MiR06) at 30°C and 37°C = 0.92
- pH of MiR05/06: 7.2 (20°C), 7.2 (25°C), 7.1 (30°C), 7.1 (35°C), 7.0 (37°C)
To increase oxygen levels small volumes (µl) of 200 mM H2O2 stock concentration are injected into the O2k-chamber filled with MiR06.
Preparation of MiR05 (MiR06) stock solution
- 1) weigh given amounts of the listed chemicals (except BSA and lactobionic acid) 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
- 5) adjust the pH to 7.1 with 5 N KOH at 30 °C
- 6) transfer the MiR05 stock solution to a 1000 ml volumentric glass flask and add H20 to a final volume of 1000 ml = MiR05
- 7) check pH again and adjust if necessary with small amounts of 5 N KOH
- 8) dissolve the BSA in a subsample of the MiR05 stock solution and add to the final MiR05 (the separate preparation of the BSA solution is recommended, since BSA produces foams that do not dissolve easily)
- 9) to prepare MiR06, add 280 000 units of catalase (100 mg of catalase powder containing 2800 u/mg solid)
- 10) divide into 40 ml portions and store at -20 °C in plastic vials
- MiR06 can also be prepared by adding 5 µl of the catalase stock solution directly into the O2k-chamber filled with MiR05 at the start of the experiment. The final catalase concentration in the 2 ml Oxygraph-2k chamber = 280 u/ml.
Chemicals for MiR05/MiR06
Caution: Chemicals stored in the fridge or freezer should be allowed to reach 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|
|Lactobionic acid||60 mM||358.3||120 ml of 0.5 M K-lactobionate stock||Aldrich 153516 or Sigma L2398, 100 g, store at R.T.|
Preparation of K-lactobionate stock solution
- 1) weigh 35.83 g lactobionic acid into a 250 ml glass beaker
- 2) add 100 ml H2O and dissolve by stirring on magnetic stirrer
- 2) check pH (is approx. 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, 2000-5000 units*/mg, Sigma C 9322, store at -20 °C
- Stock solution: 112000 u/ml (dissolved in MiR05)
- Example: 'Catalase lypophilized powder, 2800 units/mg solid and 3500 units/mg protein'
- 1) Use 'units/mg solid' for your calculations
- 2) Result: 40 mg catalase powder (2800 u/mg) are dissolved in 1 ml MiR05 to obtain a catalase stock solution with 112000 u/ml.
- 3) Titrate 5 µl of the catalase stock solution into the 2 ml chamber to achieve a final concentration in the chamber of 280 u*/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 A240. '
Preparation of 200 mM H2O2 stock solution
H2O2: Hydrogen peroxide solution, 50 wt. % in H2O, stabilized, Sigma 516813, store in the fridge, please see e.g. this link for handling and savety instructions concerning hydrogen peroxide.
- 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 plastic vial in aluminium foil (solution is light sensitive) and store in the fridge
- 4) during experiments keep the solution on ice
titration of 3 µl of H2O2 into 2 ml chamber would increase the concentration of O2 by approx. 150 nmol/ml
For detailed information on chemicals see MiPNet14.13.
Preparation of MiR07
- 1) prepare every morning fresh by adding 3 mg/mL creatine monohydrate (Fluka 27900) to MiR06
- 2) stirr gently on a magnetic stirrer