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__TOC__ == Application in [[HRR]] == ::: '''P: Pyruvate''' (pyruvic acid, sodium salt, C<sub>3</sub>H<sub>3</sub>O<sub>3</sub>Na); Sigma P 2256, 25 g, store at 4 °C; M = 110.0 g·mol<sup>-1</sup> :::: It is possible to weigh the powder beforehand in the Eppendorf-type tubes and store these tubes at 4 °C, to be diluted only on the day of use. :::: '''Preparation of 2 M stock solution''' (dissolved in H<sub>2</sub>O) for use in '''2-mL O2k-chamber''': ::::# Prepare fresh everyday. ::::# Weigh 44 mg of pyruvic acid directly into a 0.5 mL Eppendorf tube. ::::# Add 0.2 mL H<sub>2</sub>O. ::::# Adjust pH to 7.0. :::» '''O2k manual titrations''' [[MiPNet09.12 O2k-Titrations]] ::::* Titration volume ('''2-mL O2k-chamber'''): 5 µL using a 25 µL Hamilton syringe. ::::* Final concentration: 5 mM. :::: '''Preparation of 2.5 M stock solution''' (dissolved in H<sub>2</sub>O) for use in '''0.5-mL O2k-chamber''': ::::# Prepare fresh everyday. ::::# Weigh 55 mg of pyruvic acid directly into a 0.5 mL Eppendorf tube. ::::# Add 0.2 mL H<sub>2</sub>O. ::::# Adjust pH to 7.0. :::» '''O2k manual titrations''' [[MiPNet09.12 O2k-Titrations]] ::::* Titration volume ('''0.5-mL O2k-chamber'''): 1 µL using a 10 µL Hamilton syringe. ::::* Final concentration: 5 mM. == Troubleshooting == === Unstable respiration while using pyruvate as the only substrate === :::* '''Customer ID''': [https://www.bioblast.at/index.php/AU_Melbourne_White_C AU Melbourne White C] ::::'''Question:''' :::: I am evaluating mitochondrial respiration from ''Drosophila melanogaster'' using Pyruvate, ADP, and Cytochrome C. However, I do not achieve a steady state level in OXPHOS. :::: Any advice would be appreciated. The data is attached (2019-07-17). [[File:Ticket2019072631000015.png|700px|center]] :::* <div>'''Answer:'''</div><div>Pyruvate alone is not sufficient to support NADH-linked respiration. In order to do so you need to combine pyruvate with at least a second NADH-linked substrate (e.g. Malate) or use a more complex combination of substrates (''e.g.,'' Pyruvate&Glutamate&Malate). See Fig. 5.9. in [https://wiki.oroboros.at/images/f/fc/Gnaiger_2014_Mitochondr_Physiol_Network_MitoPathways.pdf Gnaiger 2020 BEC MitoPathways] :::: Additionally, you may consult some of the publications from Drosophila melanogaster mitochondria: [[O2k-Publications:_Drosophila]]
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