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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]]. MiR06 = MiR05 plus [[catalase]]. MiR06Cr = MiR06 plus [[creatine]]. |
| |info=[[MiPNet14.13]]; [[Gnaiger 2000 MitoInTheCold]] | | |info=[[MiPNet14.13 Medium-MiR06]] |
| |type=Chemicals | | |type=Chemicals |
| }} | | }} |
| {{Labeling | | {{MitoPedia concepts|type=Chemicals |
| |instruments=Chemicals; Media | | }} |
| | {{MitoPedia methods|type=Chemicals |
| | }} |
| | {{MitoPedia O2k and high-resolution respirometry|type=Chemicals |
| | }} |
| | {{MitoPedia topics |
| | |mitopedia topic=Medium |
| |type=Chemicals | | |type=Chemicals |
| }} | | }} |
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| == Application in [[HRR]] ==
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| '''MiR06: Mitochondrial Respiration Medium - MiR06''' (MiR06 = [[#Preparation of MiR05 stock solution|MiR05]] + Catalase). Total volume = 1 litre.
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| <span style="color:#8B008B"> '''Caution:''' Chemicals stored in the fridge or freezer should be adjusted to room temperature before opening.</span>
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| ===Preparation of MiR05 stock solution===
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| ::1) weigh given amounts of the [[#Chemicals for MiR05|listed chemicals]] (except K-lactobionate) 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 the powder by stirring on a magnetic stirrer at ~30 °C
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| ::4) add 120 ml of [[#Preparation of K-lactobionate stock solution|K-lactobionate stock solution]]
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| ::5a) adjust the pH to 7.1 with 5 N KOH at 30 °C '''= MiR05'''
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| ::5b) to prepare '''MiR06''' add [[#Preparation of catalase stock stolution| catalase (final conc. 280 u*/ml)]] directly to the MiR05 stock solution
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| ::6) divide into 20 ml portions and store at -20 °C in plastic vials
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| ====Preparation of catalase stock stolution====
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| '''Addition of catalase to MiR05 stock solution of 1000 ml final volume:'''
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| '''Addition of catalse stock solution into the 2 ml oxygraph chamber:'''
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| ====Chemicals for MiR05====
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| {|border="1" cellpadding="5" 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 °C
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| |-
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| |align="right"|K-lactobionate
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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 P 153516, 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 K-lactobionate directly 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. around 2.0) and neutralize with [[#Preparation of 5 N KOH stock solution|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
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| ====Preparation of 5 N KOH stock solution====
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| '''KOH''' (Potassium hydroxide, ≥85% KOH basis, pellets); Aldrich P 1767, 1000 g, store at R.T.: FW = 56.11
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| __NOTOC__
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