ADP: Difference between revisions
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== Application in [[HRR]] == | == Application in [[HRR]] == | ||
'''D: ADP''' (Adenosine 5'diphosphate potassium salt, C<sub>10</sub>H<sub>15</sub>N<sub>5</sub>O<sub>10</sub>P<sub>2</sub>K); Sigma A 5285, 1 g, store at -20 Β°C; FW = 501.3. Alternative source | '''D: ADP''' (Adenosine 5'diphosphate potassium salt, C<sub>10</sub>H<sub>15</sub>N<sub>5</sub>O<sub>10</sub>P<sub>2</sub>K); Sigma A 5285, 1 g, store at -20 Β°C; FW = 501.3. | ||
Β | |||
'''Alternative source:''' (has been recommended for low ATP impurities and used in Oroboros Lab): MerckΒ 117105-1GM (Calbiochem; FW = 501.3; store at 4Β°C). | |||
<span style="color:#8B008B"> '''Caution:''' Chemicals stored in the fridge or freezer should be allowed to reach room temperature before opening.</span> | <span style="color:#8B008B"> '''Caution:''' Chemicals stored in the fridge or freezer should be allowed to reach room temperature before opening.</span> |
Revision as of 15:34, 22 October 2013
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 Β°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 4Β°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 Β΅l)! 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 Β°C
Preparation of 500 mM ADP stock solution with 3 mM free Mg2+:
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) 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 MgCl2/mol ADP) seperately and perform a titration of both solutions in immediate succession.
Oxygraph-2k Manual Titrations: MiPNet09.12
- Titration volume: 4-20 Β΅l using a 25 Β΅l syringe
- Final conc. in 2 ml O2k-chamber: 1-5 mM