ATP: Difference between revisions
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::2) follow steps 4-6 of the above instructions | ::2) follow steps 4-6 of the above instructions | ||
::'''Comment''': In some cases it was observed, that after defreezing ofย the 'ATP-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 ofย the 'ATP-Mg' a white precipitate has formed again. If this is the case we recommend to prepare the '''500 mM ATP stock solution''' and a MgCl<sub>2</sub> solution (0.8 mol MgCl<sub>2</sub>/mol ATP) seperately and perform a titration of both solutions in immediate succession. | ||
Revision as of 11:29, 16 May 2012
Description
Adenosine 5โ-triphosphate disodium salt hydrate (T), C10H14N5O13P3Na2.
Abbreviation: T
Reference: MiPNet03.02; MiPNet09.12
MitoPedia topics:
Substrate and metabolite
Application in HRR
T: ATP (Adenosine 5'-triphosphate disodium salt hydrate, C10H14N5O13P3Na2* (H2O)x); Sigma A 2383, 5 g, store at R.T.): FW = 614.1 containing 3.5 mol H2O/mol (FWanhydrous basis = 551.1). Alternative source: Merck 1191-1GM (Calbiochem, Adenosine 5โฒ-Triphosphate Disodium Salt; FW = 551.2; store at -20 ยฐC).
Caution: Chemicals stored in the fridge or freezer should be allowed to reach room temperature before opening.
Preparation of 500 mM ATP stock solution (dissolved in H2O):
- 1) weigh 614.1 mg of ATP (FW = 614.1 containing 3.5 mol H2O/mol)
- 2) add 1.2 ml H2O
- 3) neutralize with 5 N KOH (approx. 400 ยตl) - ATP 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 ยฐC
Preparation of 500 mM ATP stock solution with 3 mM free Mg2+:
To keep free [MgMg2+] constant during respiration measurement mix ATP with MgCl2 (0.8 mol MgCl2 /mol ATP).
MgCl2 (Scharlau MA0036: MgCl2.6H2O, FW = 203.3)
- 1) add 162.64 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 'ATP-Mg' a white precipitate has formed again. If this is the case we recommend to prepare the 500 mM ATP stock solution and a MgCl2 solution (0.8 mol MgCl2/mol ATP) seperately and perform a titration of both solutions in immediate succession.
Oxygraph-2k Manual Titrations: MiPNet09.12
- Titration volume: 4-20 ยตl using a 25 ยตl syringe
- Final conc. in 2 ml O2k-chamber: 1-5 mM