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Difference between revisions of "MiR06"

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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
}}
}}
{{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
}}
}}
== Application in [[HRR]] ==
'''MiR06: Mitochondrial Respiration Medium''' ([[#Preparation of MiR05 (MiR06) stock solution|MiR06]] = MiR05 + Catalase). Total volume = 1 litre.
::Oxygen solubility factor in MiR06 (MiR06) at 30Β°C and 37Β°C = 0.92
'''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.
===Preparation of MiR05 (MiR06) stock solution===
::1) weigh given amounts of the [[#Chemicals for MiR05|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 H<sub>2</sub>O and dissolve on a magnetic stirrer at ~30 Β°C
::4) add 120 ml of [[#Preparation of K-lactobionate stock solution|K-lactobionate stock solution]]
::5) adjust the pH to 7.1 with 5 N KOH at 30 Β°C
::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>
::7 check pH again and adjust if necessary with small amounts of 5 N KOH
::8) to prepare <span style="color:#2E8B57"> '''MiR06''' </span>, add 2.5 ml of the [[#Preparation of catalase stock solution| catalase stock solution]] to the MiR05 (take the 2.5 ml for dissolving the calculated amount of catalase powder from the just prepared MiR05 stock solution)
::9) divide into 20 ml portions and store at -20 Β°C in plastic vials
::<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.
====Chemicals for MiR05====
<span style="color:#8B008B"> '''Caution:''' Chemicals stored in the fridge or freezer should be allowed to reach room temperature before opening.</span>
{|border="1" cellpadding="3" cellspacing="0"
|+
!Compound
!Final conc.
!FW
!Addition to 1 litre final volume
!Company, product code and storage
|-
|align="right"|EGTA
|align="right"|0.5 mM
|align="right"|380.4
|align="right"|0.190 g
|align="right"|Sigma E 4378, 25 g, store at R.T.
|-
|align="right"|MgCl<sub>2</sub>.6H<sub>2</sub>0
|align="right"|3 mM
|align="right"|203.3
|align="right"|0.610 g
|align="right"|Scharlau MA 0036, 250 g, store at R.T.
|-
|align="right"|Taurine
|align="right"|20 mM
|align="right"|125.1
|align="right"|2.502 g
|align="right"|Sigma T 0625, 25 g, store at R.T.
|-
|align="right"|KH<sub>2</sub>PO<sub>4</sub>
|align="right"|10 mM
|align="right"|136.1
|align="right"|1.361 g
|align="right"|Merck 104873, 1000 g, store at R.T.
|-
|align="right"|HEPES
|align="right"|20 mM
|align="right"|238.3
|align="right"|4.77 g
|align="right"|Sigma H 7523, 250 g, store at R.T.
|-
|align="right"|D-Sucrose
|align="right"|110 mM
|align="right"|342.3
|align="right"|37.65 g
|align="right"|Roth, 4621.1, 1000 g, store at R.T.
|-
|align="right"|BSA essentially fatty acid free
|align="right"|1 g/l
|align="right"|
|align="right"|1 g
|align="right"|Sigma A 6003 fraction V, 25 g, store at 2-8 Β°C
|-
|align="right"|Lactobionic acid
|align="right"|60 mM
|align="right"|358.3
|align="right"|120 ml of 0.5 M [[#Preparation of K-lactobionate stock solution|K-lactobionate stock]]
|align="right"|Aldrich 153516 or Sigma L2398, 100 g, store at R.T.
|-
|}
====Preparation of K-lactobionate stock solution====
::1) weigh 35.83 g lactobionic acid directly into a 250 ml glass beaker
::2) add 100 ml H<sub>2</sub>O and dissolve by stirring on magnetic stirrer
::2) check pH (is approx. around 2.0) and neutralize with 5 N KOH
::4) adjust final volume to 200 ml with H<sub>2</sub>O. 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 Β°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:Β  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 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 AH<sub>240</sub>.'' '
===Preparation of 200 mM H2O2 stock solution===
'''H<sub>2</sub>O<sub>2</sub>''': Hydrogen peroxide solution, 50 wt. % in H2O, stabilized, Sigma 516813, store in the fridge
::1) pipette 114 Β΅l 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
::4) during experiments keep the solution on ice to prevent from bubble forming
'''For detailed information on chemicals see [http://www.oroboros.at/index.php?id=protocols_miro6 MiPNet14.13].'''

Latest revision as of 15:23, 8 February 2016


high-resolution terminology - matching measurements at high-resolution


MiR06

Description

Mitochondrial respiration medium, MiR06, developed for oxygraph incubations of mitochondrial preparations. MiR06 = MiR05 plus catalase. MiR06Cr = MiR06 plus creatine.

Abbreviation: MiR06

Reference: MiPNet14.13 Medium-MiR06






MitoPedia topics: Media for respirometry