Difference between revisions of "ADP"
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::'''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 | ::'''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 MgCl<sub>2</sub> solution (0.6 mol MgCl<sub>2</sub>/mol ADP) seperately and perform a titration of both solutions in immediate succession. | ||
'''Oxygraph-2k | '''Oxygraph-2k manual titrations''' [[MiPNet09.12 O2k-Titrations]] | ||
::* Titration volume: 4-20 µl using a 25 µl syringe | ::* Titration volume: 4-20 µl using a 25 µl syringe (2 ml O2k-chamber). | ||
::* Final | ::* Final concentration: 1-5 mM. |
Revision as of 04:57, 16 April 2014
Description
Adenosine diphosphate is a nucleotid and a substrate of ANT and ATP synthase. ADP is the discharged or energy-poor counterpart of ATP. ADP can accept chemical energy by regaining a phosphate group to become ATP, at the expense of either solar energy (in photosynthetic cells) or chemical energy (in heterotrophic cells).
Abbreviation: D
Reference: MiPNet03.02, MiPNet09.12
MitoPedia methods:
Respirometry
MitoPedia topics:
Substrate and metabolite
Application in HRR
D: ADP (Adenosine 5'diphosphate potassium salt, C10H15N5O10P2K); Sigma A 5285, 1 g, store at -20 °C; FW = 501.3.
Alternative source: (has been recommended for low ATP impurities and used in Oroboros Lab): Merck 117105-1GM (Calbiochem; FW = 501.3; store at 4°C).
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!
- 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+ (dissolved in H2O):
To keep free [Mg2+] constant during respiration measurement mix ADP with MgCl2 (0.6 mol MgCl2/mol ADP).
MgCl2 (Scharlau MA0036: MgCl2.6H2O, FW = 203.3)
- 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!
- 4) add 121,98 mg MgCl2 .White precipitate will occur, which will be dissolved after 1-2 min of stirring on magnetic stirrer at room temperature!
- 5) check pH and adjust to 7 if necessary
- 6) adjust final volume to 2 ml and divide into 0.2 ml portions
- 7) store frozen at -80 °C
- 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 MgCl2/mol ADP) seperately and perform a titration of both solutions in immediate succession.
Oxygraph-2k manual titrations MiPNet09.12 O2k-Titrations
- Titration volume: 4-20 µl using a 25 µl syringe (2 ml O2k-chamber).
- Final concentration: 1-5 mM.