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| == Problem ==
| | #REDIRECT [[MitoPedia:_Respiratory_control_ratios]] |
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| At a high mitochondrial concentration (0.5 mg protein/ml), ADP is exhausted very rapidly if added at a concentration typically used for [[State 3]] measurements (200-300 µM), which may stimulate flux below the ADP-saturated state of [[OXPHOS capacity]]. The slope increases sharply and returns to [[State 4]] ([[LEAK]]), even before the plot of flux shows the true maximum, resulting in a low apparent '''[[RCR]]'''.
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| == Tests and Solutions ==
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| # Reduce the data recording interval (standard is 2 seconds) to the minimum of 0.2 s (setting in the Oxygraph Control window; see [[MiPNet12.06 O2k-Start]]). As the data recording interval is reduced, the flux appears more noisy, but represents transitions more accurately and reduces the apparent time-delay. At high flux per volume, the increased noise level presents no problem. Flux then shows a period of constant OXPHOS capacity rather than a sharp peak. | |
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| # Reduce the mitochondrial concentration, thus prolonging the duration of ADP-saturated respiration (OXPHOS capacity) over 120 seconds. If flux reaches a constant maximum value, then analysis is possible without application of signal deconvolution (standard plot of flux in DatLab).
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| # Otherwise, export the data into [http://www.oroboros.at/index.php?o2k-datlab2 DatLab 2], and apply a time correction (signal deconvolution), as described by [[Gnaiger 2001 Respir Physiol]].
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| # See also ‘Notes on [[Time resolution]]’.
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| {{#set:Scientific service=Isolated Mitochondria | Scientific service=Respiratory States}}
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| __SHOWFACTBOX__
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| {{Troubleshooting}}
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