Quantification of Extracellular Metabolites
Corresponding Organization : Instituto de Bioquímica Vegetal y Fotosíntesis
Variable analysis
- None explicitly mentioned
- Concentration of pyruvate
- Concentration of 2-oxoglutarate
- Culture samples centrifuged at 15,000 g for 10 min at 4 °C
- Supernatants quick-frozen in liquid nitrogen, lyophilized, and stored at -20 °C
- Samples resuspended in 600 µl of H2O
- Enzyme-coupled assays performed in 375 mM Tris–HCl (pH 7.5) with 0.11 mM NADH
- 50 mM NH4Cl used for 2-oxoglutarate quantification
- Reaction triggered by addition of lactate dehydrogenase or glutamate dehydrogenase
- NADH oxidation measured spectrophotometrically at 340 nm
- Standard curves of known amounts of pyruvate and 2-oxoglutarate
- None explicitly mentioned
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