|
Β |
(21 intermediate revisions by 3 users not shown) |
Line 1: |
Line 1: |
| {{MitoPedia | | {{MitoPedia |
| |abbr=MiR06 | | |abbr=MiR06 |
| |description='''Mitochondrial respiration medium, MiR06''', developed for oxygraph incubations of [[mitochondrial preparations]].Β MiR06 = [[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 Life in the Cold]] | | |info=[[MiPNet14.13 Medium-MiR06]] |
| |type=Chemicals | | |type=Chemicals |
| }} | | }} |
| {{MitoPedia methods | | {{MitoPedia concepts|type=Chemicals |
| |mitopedia method=Respirometry | | }} |
| |type=Chemicals | | {{MitoPedia methods|type=Chemicals |
| | }} |
| | {{MitoPedia O2k and high-resolution respirometry|type=Chemicals |
| }} | | }} |
| {{MitoPedia topics | | {{MitoPedia topics |
Line 13: |
Line 15: |
| |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
| |
|
| |
| ::pH of MiR05/06: 7.2 (20Β°C), 7.2 (25Β°C), 7.1 (30Β°C), 7.1 (35Β°C), 7.0 (37Β°C)
| |
|
| |
| '''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 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 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) 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 <span style="color:#2E8B57"> '''MiR06'''</span>, 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 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/MiR06 ====
| |
|
| |
| <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 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. 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.
| |
| ::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.
| |
|
| |
| '''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>.'' '
| |
|
| |
|
| |
| === 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, 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.pdfΒ this link] for handling and savety instructions concerning hydrogen peroxide.
| |
|
| |
| ::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 aluminium foil (solution is light sensitive) and store in the fridge
| |
| ::4) during experiments keep the solution on ice
| |
|
| |
| '''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
| |
|
| |
| '''For detailed information on chemicals see [http://www.oroboros.at/index.php?protocols_miro6 MiPNet14.13].'''
| |
|
| |
| === Preparation of MiR07 ===
| |
| 1)add in fresh 3 mg/mL creatine monohydrate (Sigma 27900) in MiR06
| |
| 2)stirring gently on magnetic stirrer
| |