Difference between revisions of "ADP"

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::1) weigh 501.3 mg of ADP (FW = 501.3) = (1 mmol ADP)
 
::1) weigh 501.3 mg of ADP (FW = 501.3) = (1 mmol ADP)
::2) add 1.2 ml H<sub>2</sub>O  (ADP is not dissolved at this stagw.
+
::2) add 1.2 ml H<sub>2</sub>O  (ADP is not dissolved at this stage.
 
::3) neutralize with 5 N KOH (approx. 450 µl)  !ADP will dissolve after addition of KOH!  (this is '''Solution A''')
 
::3) neutralize with 5 N KOH (approx. 450 µl)  !ADP will dissolve after addition of KOH!  (this is '''Solution A''')
 
::4) check pH and adjust to 7 if necessary  
 
::4) check pH and adjust to 7 if necessary  

Revision as of 13:26, 5 December 2011

Bioblasts - Richard Altmann and MiPArt by Odra Noel
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MitoPedia

ADP

Description

Adenosine 5’diphosphate (D), C10H15N5O10P2K, potassium salt; substrate of ANT and ATP synthase.

Abbreviation: D

Reference: MiPNet03.02, MiPNet09.12


MitoPedia methods: Respirometry 


MitoPedia topics: Substrate and metabolite 

Application in HRR

D: ADP (Adenosine 5'diphosphate, C10H15N5O10P2K, potassium salt); Sigma A 5285, 1 g, store at -20 °C): FW = 501.3. Alternative source, that has been recommended for low ATP impurities: Merck 117105-1 (Calbiochem).

Caution: Chemicals stored in the fridge or freezer should be allowed to reach room temperature before opening.


Preparation of 500 mM ADP stock solution (dissolved in H2O):

1) weigh 501.3 mg of ADP (FW = 501.3) = (1 mmol ADP)
2) add 1.2 ml H2O (ADP is not dissolved at this stage.
3) neutralize with 5 N KOH (approx. 450 µl)  !ADP will dissolve after addition of KOH! (this is Solution A)
4) check pH and adjust to 7 if necessary
5) adjust final volume to 2 ml and divide into 0.2 ml portions
6) store frozen at -80 °C


Preparation of 500 mM ADP stock solution with 3 mM free Mg2+:

To keep free [MgMg2+] constant during respiration measurement mix ADP (or ATP) with MgCl2 (0.6 mol/mol ADP or 0.8 mol/mol ATP).

MgCl2 (Scharlau MA0036: MgCl2.6H2O, FW = 203.3)

1) add 121,98 mg MgCl2 to Solution A !White precipitate will occur, which will be dissolved after 1-2 min of stirring on magnetic stirrer at room temperature!
2) follow steps 4-6 of the above instructions
Comment: In some cases it was observed, that after defreezing of the 'ADP-Mg' a white precipitate has formed again. If this is the case we recommend to prepare the ADP stock solution and a MgCl2 solution (0.6 mol/mol ADP or 0.8 mol/mol ATP) seperately and perform a titration of both solutions in immediate succession.


Oxygraph-2k Manual Titrations: MiPNet09.12

  • Titration volume: 8-20 µl using a 25 µl syringe
  • Final conc. in 2 ml O2k-chamber: 2-5 mM