Difference between revisions of "Talk:Respiratory acceptor control ratio"
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<big>'''Tests and solutions'''</big> | <big>'''Tests and solutions'''</big> | ||
# Reduce the data recording interval (standard is 2 seconds) to the minimum of 0.2 s (setting in the O2k control window; see [[MiPNet19.18A O2k- | # Reduce the data recording interval (standard is 2 seconds) to the minimum of 0.2 s (setting in the O2k control window; see [[MiPNet19.18A 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. | ||
# 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). | # 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). | ||
# Otherwise, export the data into [http://www.oroboros.at/?O2k-datlab2 DatLab 2], and apply a time correction (signal deconvolution), as described by [[Gnaiger 2001 Respir Physiol]]. | # Otherwise, export the data into [http://www.oroboros.at/?O2k-datlab2 DatLab 2], and apply a time correction (signal deconvolution), as described by [[Gnaiger 2001 Respir Physiol]]. | ||
# See also ‘Notes on [[Time resolution]]’. | # See also ‘Notes on [[Time resolution]]’. | ||
Revision as of 10:18, 14 August 2016
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Problem: The RCR is low in isolated mitochondria
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.
Tests and solutions
- Reduce the data recording interval (standard is 2 seconds) to the minimum of 0.2 s (setting in the O2k control window; see MiPNet19.18A 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.
- 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).
- Otherwise, export the data into DatLab 2, and apply a time correction (signal deconvolution), as described by Gnaiger 2001 Respir Physiol.
- See also ‘Notes on Time resolution’.
