Difference between revisions of "MiR06"

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=== Preparation of MiR07 ===
 
=== Preparation of MiR07 ===
::1) add in fresh 3 mg/mL creatine monohydrate (Fluka 27900) in MiR06
+
::1) prepare every morning fresh by adding 3 mg/mL creatine monohydrate (Fluka 27900) to [[MiR06]]
::2) stirring gently on magnetic stirrer
+
::2) stirr gently on a magnetic stirrer

Revision as of 08:11, 30 November 2011

Bioblasts - Richard Altmann and MiPArt by Odra Noel
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MitoPedia

MiR06

Description

Mitochondrial respiration medium, MiR06, developed for oxygraph incubations of mitochondrial preparations. MiR06 = MiR05 plus catalase.

Abbreviation: MiR06

Reference: MiPNet14.13; Gnaiger 2000 Life in the Cold


MitoPedia methods: Respirometry 


MitoPedia topics: Media for respirometry 

Application in HRR

MiR06: Mitochondrial Respiration Medium (MiR06 = MiR05 + Catalase). Total volume = 1 litre.

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