Difference between revisions of "MiR06"

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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 = MiR05 + Catalase). Total volume = 1 litre.

Oxygen solubility factor in MiR06 (MiR06) at 30°C and 37°C = 0.92

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 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) to prepare MiR06 , add 280 000 units of catalase (100 mg of catalase powder containing 2800 u/mg solid)

9) 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

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.

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

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: 3 µl of H2O2 into 2 ml chamber would increase the concentration of O2 for around 150 nmol/ml

For detailed information on chemicals see MiPNet14.13.

@@ Line 117: / Line 117: @@
 ::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.
-'''Unit definition:''' '''* Units''' of enzymatic activitiy (u) in µmol/min; assay used by Sigma Aldrich: ' ''One unit will decompose 1.0 μmole of H<sub>2</sub>O<sub>2</sub>  per min at pH 7.0 at 25 °C, while the H<sub>2</sub>O<sub>2</sub> concentration falls from 10.3 to 9.2 mM, measured by the rate of decrease of AH<sub>240</sub>.'' '
+'''Unit definition:''' '''* Units''' of enzymatic activitiy (u) in µmol/min; assay used by Sigma Aldrich: ' ''One unit will decompose 1.0 μmole of H<sub>2</sub>O<sub>2</sub>  per min at pH 7.0 at 25 °C, while the H<sub>2</sub>O<sub>2</sub> concentration falls from 10.3 to 9.2 mM, measured by the rate of decrease of A<sub>240</sub>.'' '
@@ Line 126: / Line 126: @@
 ::1) pipette 114 µl of 17.6 M H<sub>2</sub>O<sub>2</sub> into 10 ml plastic vial
 ::2) add H<sub>2</sub>O to a total volume of 10 ml
-::3) wrap plastic vial in tin foil (solution is light sensitive) and store in the fridge
+::3) wrap plastic vial in aluminium foil (solution is light sensitive) and store in the fridge
-::4) during experiments keep the solution on ice to prevent from bubble forming
+::4) during experiments keep the solution on ice
+titration: 3 µl of H2O2 into 2 ml chamber would increase the concentration of O2 for around 150 nmol/ml
 '''For detailed information on chemicals see [http://www.oroboros.at/index.php?id=protocols_miro6 MiPNet14.13].'''