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'''C''' [[MiPNet18.13 IOC84 Alaska]]  +
'''C''' [[MiPNet18.13 IOC84 Alaska]]  +
'''B''' [[Lemieux 2011 Int J Biochem Cell Biol]]  +
2
[[Proton_flux]], [[O2k-pH_ISE-Module]]  +
[[Jezek 2020 Antioxid Redox Signal]]  +
[http://www.ncbi.nlm.nih.gov/pubmed/7317017 Bauché 1981 Biochem J], [[Osiki 2016 FASEB J]]  +
A
[[MiPNet03.02]], [[MiPNet09.12]] __TOC__ == 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), ::: Merck 117105-1GM ('''Calbiochem''') M = 501.3 g·mol<sup>-1</sup>; store at 4 °C. Recommended due to lower ATP impurities, we use this ADP for preparation of 'ADP-Mg' solution in [[AT_Innsbruck_Oroboros |Oroboros Lab]]. ::: Alternative source: Sigma A 5285, 1 g, store at -20 °C; M = 501.3 g·mol<sup>-1</sup>. :::: To keep free [Mg<sup>2+</sup>] constant during respiration measurement in [[MiR06]] or MiR05, mix ADP with MgCl<sub>2</sub> (0.6 mol MgCl<sub>2</sub>/mol ADP). :::: To find out more about the importance of adding Mg<sup>2+</sup>, please refer to [https://en.wikipedia.org/wiki/Magnesium_in_biology#Essential_role_in_the_biological_activity_of_ATP here] :::: '''MgCl<sub>2</sub>''' (Scharlau MA0036: MgCl<sub>2</sub>.6H<sub>2</sub>O, M = 203.3 g·mol<sup>-1</sup>) :::: For instructions without MgCl<sub>2</sub> and with smaller amounts of ADP see [[Talk:ADP]] :::: <span style="color:#8B008B"> '''Caution:''' Chemicals stored in the fridge or freezer should be allowed to reach room temperature before opening.</span> :::: '''Preparation of 500 mM ADP stock solution with 300 mM free Mg<sup>2+</sup>''' (dissolved in H<sub>2</sub>O): ::::# Take 1 g of ADP (can be orderd in portions of 1 g per vial). ::::# Add 2.3 mL H<sub>2</sub>O (ADP is not dissolved at this stage). ::::# Neutralize with 5 M KOH (approx. 900 µl). ADP will dissolve after addition of KOH. ::::# Add 243.96 mg MgCl<sub>2</sub>. White precipitate will occur, which will dissolve during 1-2 min stirring or shaking. ::::# Check pH and adjust to 7 with 5 M KOH if necessary (usually about 30 to 50 µl). Addition of KOH causes precipitation. Carefully stir or shake it until the solution is clear again, then add the next droplet of KOH. ::::# Adjust final volume to 4 mL and divide into 0.2 mL portions. ::::# Store at -20 °C. :::: '''Comment''': In some cases, a white precipitate was observed after thawing the 'ADP-Mg' solution. 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) separately and perform a titration of both solutions in immediate succession. :::: '''Preparation of 200 mM ADP stock solution without Mg<sup>2+</sup> for experiments with [[Magnesium Green]]''' (dissolved in H<sub>2</sub>O): :::» '''O2k manual titrations''' [[MiPNet09.12 O2k-Titrations]] :::: '''500 mM ADP:''' ::::* Titration volume ('''2-mL O2k-chamber'''): 4-20 µL using a 25 µL Hamilton syringe. ::::* Titration volume ('''0.5-mL O2k-chamber'''): 1-5 µL using a 10 µL Hamilton syringe. ::::* Final concentration: 1-5 mM. :::: '''200 mM ADP for experiments with [[Magnesium Green]]:''' ::::* Titration volume ('''2-mL O2k-chamber'''): 10-50 µL using a 50 µL Hamilton syringe. ::::* Final concentration: 1-5 mM. == DatLab oxygen flux: performance and data analysis == ::::* Flux increases to OXPHOS capacity at kinetically saturating [ADP]. The full activation of ADP may require time, however for the evaluation of OXPHOS capacity it is needed to reach the steady state. ::::* Recommendation: use MgADP2+. ::::* Recommendation: 2.5 mM final conc, 10 µL titration. Information on optimal [ADP]: [[Gnaiger_2001_Respir_Physiol]] ::::* Recommendation for permeabilized fibers: 5 mM or higher final conc. Information on optimal [ADP]: [[Gnaiger_2001_Respir_Physiol]], [[Permeabilized muscle fibers]]. ::::* Guidelines for performing and evaluating respirometric assays: [[DatLab_oxygen_flux:_performance_and_data_analysis]] == ADP dependence of respiration == :::: The assumption of linearity (linear regression of oxygen concentration over time) is frequently not valid for various reasons other than [[oxygen kinetics]]. In classical ‘State 3’, ADP levels are ‘high’ ([[Chance_1955_JBC-III|Chance and Williams, 1955]]), but not necessarily saturating ([[MitoPedia: Respiratory states]]). Then, an ADP-dependent decline of respiration is observed immediately after titration of a sub-saturating concentration of ADP, which is obscured by any linear regression. This has caused in the past a tremendous underestimation of the apparent Km for ADP, perpetuated even today with the uncritical application of non-adequate software implementing the simple linearity approach only. For a critical approach to ADP kinetics, see [[Gnaiger_2000_Proc_Natl_Acad_Sci_U S A|Gnaiger et al 2000]] and [[Gnaiger_2001_Respir_Physiol|Gnaiger 2001]]. [[File:Questions.jpg|left|40px]] <br/> <div class="toccolours mw-collapsible mw-collapsed"> ::: <span style="font-size:105%; color:##424242">'''» Keywords'''</span> <div class="mw-collapsible-content"> ::: '''Specific''' ::::» [[ANT]] ::::» [[OXPHOS]] ::::» [[OXPHOS capacity]] ::::» [[Phosphorylation system]] ::: '''General''' ::::» [[ATP]] ::::» [[ATP synthase]] ::::» [[MiR06]] ::::» [[Mitochondrial preparations]] ::::» [[MiPNet09.12 O2k-Titrations|O2k-Titrations]] </div> </div> <br/> == References == <table class="sortable wikitable smwtable broadtable" width="100%"><th class="Bioblast-link">Bioblast link</th><th class="Reference">[[:Property:Has title|Reference]]</th><th class="Year">[[:Property:Was published in year|Year]]</th><tr data-row-number="1" class="row-odd"><td class="Bioblast-link smwtype_wpg">[[:Davis 1980 Eur J Biochem|Davis 1980 Eur J Biochem]]</td><td class="Reference smwtype_txt">Davis EJ, Spydevold O, Bremer J (1980) Pyruvate carboxylase and propionyl-CoA carboxylase as anaplerotic enzymes in skeletal muscle mitochondria. Eur J Biochem 110:255-62.</td><td class="Year smwtype_txt" data-sort-value="1980">1980</td></tr><tr data-row-number="2" class="row-even"><td class="Bioblast-link smwtype_wpg">[[:BEC 2020.1 doi10.26124bec2020-0001.v1|BEC 2020.1 doi10.26124bec2020-0001.v1]]</td><td class="Reference smwtype_txt">Gnaiger Erich et al ― MitoEAGLE Task Group (2020) Mitochondrial physiology. Bioenerg Commun 2020.1:44 pp. [[doi:10.26124/bec:2020-0001.v1]]</td><td class="Year smwtype_txt" data-sort-value="2020">2020</td></tr><tr data-row-number="3" class="row-odd"><td class="Bioblast-link smwtype_wpg">[[:Ost 2015 Fatty Acid Oxidation O2k-Network Discussion Forum|Ost 2015 Fatty Acid Oxidation O2k-Network Discussion Forum]]</td><td class="Reference smwtype_txt">Ost 2015 Fatty Acid Oxidation O2k-Network Discussion Forum</td><td class="Year smwtype_txt"></td></tr><tr data-row-number="4" class="row-even"><td class="Bioblast-link smwtype_wpg">[[:MiPNet03.02 Chemicals-Media|MiPNet03.02 Chemicals-Media]]</td><td class="Reference smwtype_txt">Selected media and chemicals for respirometry with mitochondrial preparations.</td><td class="Year smwtype_txt">2016-08-30</td></tr><tr data-row-number="5" class="row-odd"><td class="Bioblast-link smwtype_wpg">[[:Vajda 2009 FEBS J|Vajda 2009 FEBS J]]</td><td class="Reference smwtype_txt">Vajda S, Mándi M, Konràd C, Kiss G, Ambrus A, Adam-Vizi V, Chinopoulos C (2009) A re-evaluation of the role of matrix acidification in uncoupler-induced Ca<sup>2+</sup> release from mitochondria. FEBS J 276:2713-24.</td><td class="Year smwtype_txt" data-sort-value="2009">2009</td></tr><tr data-row-number="6" class="row-even"><td class="Bioblast-link smwtype_wpg">[[:MiPNet09.12 O2k-Titrations|MiPNet09.12 O2k-Titrations]]</td><td class="Reference smwtype_txt">[[Image:O2k-Protocols.jpg|right|80px|link=http://wiki.oroboros.at/index.php/O2k-Protocols|O2k-Protocols]] O2k manual titrations: SUIT protocols with mitochondrial preparations.</td><td class="Year smwtype_txt">2020-08-17</td></tr></table>  
[http://www.ncbi.nlm.nih.gov/pubmed/22436748 Hardie_2012_Nat Rev Mol Cell Biol]; [http://www.ncbi.nlm.nih.gov/pubmed/19584320 Steinberg_2009_Physiol Rev]  +
[https://asapbio.org/about-us asapbio.org]  +
[[MiPNet03.02 Chemicals-Media]]  +
[[Walker JE]]  +
[[LEAK respiration]]  +
https://iupac.org/  +
[[Cohen 2008 IUPAC Green Book]]  +
[[Hochachka 2002 Oxford Univ Press | Hochachka, Somero 2002]]  +
[[Hochachka 2002 Oxford Univ Press | Hochachka, Somero 2002]]  +
[[Paul 1991 Pharm Technol]]  +
[[Gnaiger 2020 BEC MitoPathways]]  +
[[Cohen 2008 IUPAC Green Book]]  +