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| {{MitoPedia | | {{MitoPedia |
| |abbr=MiR06 | | |abbr=MiR06 |
| |description='''Mitochondrial respiration medium 06''', developed for oxygraph incubations of [[mitochondrial preparations]] = MiR05 plus catalase. | | |description='''Mitochondrial respiration medium, [[MiPNet14.13 Medium-MiR06|MiR06]]''', developed for oxygraph incubations of [[mitochondrial preparations]].Β MiR06 = MiR05 plus [[catalase]]. MiR06Cr = MiR06 plus [[creatine]]. |
| |info=[[MiPNet14.13]]; [[Gnaiger 2000 MitoInTheCold]] | | |info=[[MiPNet14.13 Medium-MiR06]] |
| |type=Chemicals | | |type=Chemicals |
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| | {{MitoPedia concepts|type=Chemicals |
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| | {{MitoPedia methods|type=Chemicals |
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| | {{MitoPedia O2k and high-resolution respirometry|type=Chemicals |
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| {{MitoPedia topics | | {{MitoPedia topics |
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| |type=Chemicals | | |type=Chemicals |
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| == Application in [[HRR]] ==
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| '''MiR06: Mitochondrial Respiration Medium''' ([[#Preparation of MiR05 (MiR06) stock solution|MiR06]] = MiR05 + Catalase). Total volume = 1 litre.
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| ::Oxygen solubility factor in MiR06 (MiR06) at 30Β°C and 37Β°C = 0.92
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| ::pH of MiR05/06: 7.2 (20Β°C), 7.2 (25Β°C), 7.1 (30Β°C), 7.1 (35Β°C), 7.0 (37Β°C)
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| '''To increase oxygen levels''' small volumes (Β΅l) of [[#Preparation of 200 mM H2O2 stock solution|200 mM H<sub>2</sub>O<sub>2</sub> stock concentration]] are injected into the O2k-chamber filled with MiR06.
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| ==Preparation of MiR05 (MiR06) stock solution==
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| ::1) weigh given amounts of the [[#Chemicals for MiR05|listed chemicals]] (except BSA and lactobionic acid) and transfer to a 1000 ml glass beaker
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| ::2) disrupt big lumps mechanically. It is recommended to do this before adding water, because during dissolution these lumps do not disintegrate easily.
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| ::3) add ~800 ml H<sub>2</sub>O and dissolve on a magnetic stirrer at ~30 Β°C
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| ::4) add 120 ml of [[#Preparation of K-lactobionate stock solution|K-lactobionate stock solution]]
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| ::5) adjust the pH to 7.1 with 5 N KOH at 30 Β°C
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| ::6) transfer the MiR05 stock solution to a 1000 ml volumentric glass flask and add H20 to a final volume of 1000 ml <span style="color:#43CD80"> = '''MiR05''' </span>
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| ::7) check pH again and adjust if necessary with small amounts of 5 N KOH
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| ::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)
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| ::9) to prepare <span style="color:#2E8B57"> '''MiR06'''</span>, add 280 000 units of catalase (100 mg of catalase powder containing 2800 u/mg solid)
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| ::10) divide into 40 ml portions and store at -20 Β°C in plastic vials
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| ::<span style="color:#2E8B57"> '''MiR06''' </span> can also be prepared by adding 5 Β΅l of the [[#Preparation of catalase stock solution|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.
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| ====Chemicals for MiR05====
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| <span style="color:#8B008B"> '''Caution:''' Chemicals stored in the fridge or freezer should be allowed to reach room temperature before opening.</span>
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| {|border="1" cellpadding="3" cellspacing="0"
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| |+
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| !Compound
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| !Final conc.
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| !FW
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| !Addition to 1 litre final volume
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| !Company, product code and storage
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| |-
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| |align="right"|EGTA
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| |align="right"|0.5 mM
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| |align="right"|380.4
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| |align="right"|0.190 g
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| |align="right"|Sigma E 4378, 25 g, store at R.T.
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| |-
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| |align="right"|MgCl<sub>2</sub>.6H<sub>2</sub>0
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| |align="right"|3 mM
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| |align="right"|203.3
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| |align="right"|0.610 g
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| |align="right"|Scharlau MA 0036, 250 g, store at R.T.
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| |-
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| |align="right"|Taurine
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| |align="right"|20 mM
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| |align="right"|125.1
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| |align="right"|2.502 g
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| |align="right"|Sigma T 0625, 25 g, store at R.T.
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| |-
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| |align="right"|KH<sub>2</sub>PO<sub>4</sub>
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| |align="right"|10 mM
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| |align="right"|136.1
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| |align="right"|1.361 g
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| |align="right"|Merck 104873, 1000 g, store at R.T.
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| |-
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| |align="right"|HEPES
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| |align="right"|20 mM
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| |align="right"|238.3
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| |align="right"|4.77 g
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| |align="right"|Sigma H 7523, 250 g, store at R.T.
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| |-
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| |align="right"|D-Sucrose
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| |align="right"|110 mM
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| |align="right"|342.3
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| |align="right"|37.65 g
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| |align="right"|Roth, 4621.1, 1000 g, store at R.T.
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| |-
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| |align="right"|BSA essentially fatty acid free
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| |align="right"|1 g/l
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| |align="right"|
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| |align="right"|1 g
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| |align="right"|Sigma A 6003 fraction V, 25 g, store at 2-8 Β°C
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| |-
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| |align="right"|Lactobionic acid
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| |align="right"|60 mM
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| |align="right"|358.3
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| |align="right"|120 ml of 0.5 M [[#Preparation of K-lactobionate stock solution|K-lactobionate stock]]
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| |align="right"|Aldrich 153516 or Sigma L2398, 100 g, store at R.T.
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| |-
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| |}
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| ====Preparation of K-lactobionate stock solution====
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| ::1) weigh 35.83 g lactobionic acid into a 250 ml glass beaker
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| ::2) add 100 ml H<sub>2</sub>O and dissolve by stirring on magnetic stirrer
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| ::2) check pH (is approx. 2.0) and neutralize with 5 N KOH
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| ::4) adjust final volume to 200 ml with H<sub>2</sub>O. It is best to use a 200 ml volumetric glass flask.
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| ::5) check pH again and adjust to 7 if necessary (5 N KOH)
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| ===Preparation of catalase stock solution===
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| ::'''Catalase''' lypophilized powder, 2000-5000 '''units*'''/mg, Sigma C 9322, store at -20 Β°C
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| ::'''Stock solution:''' 112000 u/ml (dissolved in MiR05)
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| ::'''Example:''' 'Catalase lypophilized powder, 2800 units/mg solid and 3500 units/mg protein'
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| ::1) Use 'units/mg solid' for your calculations
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| ::2) Result:Β 40 mg catalase powder (2800 u/mg) are dissolved in 1 ml MiR05 to obtain a catalase stock solution with 112000 u/ml.
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| ::3) Titrate 5 Β΅l of the catalase stock solution into the 2 ml chamber to achieve a final concentration in the chamber of 280 u*/ml.
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| '''Unit definition:''' '''* Units''' of enzymatic activitiy (u) in Β΅mol/min; assay used by Sigma Aldrich: ' ''One unit will decompose 1.0 ΞΌmole of H<sub>2</sub>O<sub>2</sub>Β per min at pH 7.0 at 25 Β°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>.'' '
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| ===Preparation of 200 mM H2O2 stock solution===
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| '''H<sub>2</sub>O<sub>2</sub>''': Hydrogen peroxide solution, 50 wt. % in H2O, stabilized, Sigma 516813, store in the fridge, please see e.g. [http://www.solvayasiapacific.com/static/wma/pdf/1/8/5/8/1/Hydrogen%20Peroxide%20Handling%20%20Storage%201%28English%20version%29.pdf] for handling and savety instructions concerning hydrogen peroxide.
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| ::1) pipette 114 Β΅l of 17.6 M H<sub>2</sub>O<sub>2</sub> into 10 ml plastic vial
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| ::2) add H<sub>2</sub>O to a total volume of 10 ml
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| ::3) wrap plastic vial in aluminium foil (solution is light sensitive) and store in the fridge
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| ::4) during experiments keep the solution on ice
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| '''titration''' of 3 Β΅l of H<sub>2</sub>O<sub>2</sub> into 2 ml chamber would increase the concentration of O<sub>2</sub> by approx. 150 nmol/ml
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| '''For detailed information on chemicals see [http://www.oroboros.at/index.php?id=protocols_miro6 MiPNet14.13].'''
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