Difference between revisions of "RCR is low in isolated mitochondria"
From Bioblast
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== Problem == | == Problem == | ||
At a high mitochondrial concentration (0.5 mg protein/ml), ADP is exhausted very rapidly. The slope increases sharply and returns to state | At a high mitochondrial concentration (0.5 mg protein/ml), ADP is exhausted very rapidly. The slope increases sharply and returns to State 4 (LEAK state), even before the plot of flux shows the true maximum value of OXPHOS capacity (State 3), resulting in a low apparent RCR. | ||
== Tests and Solutions == | == Tests and Solutions == | ||
Revision as of 18:14, 13 November 2011
Problem
At a high mitochondrial concentration (0.5 mg protein/ml), ADP is exhausted very rapidly. The slope increases sharply and returns to State 4 (LEAK state), even before the plot of flux shows the true maximum value of OXPHOS capacity (State 3), 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 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 state 3 respiration rather than a sharp peak, and routine analysis is possible in DatLab 4.
- Reduce the mitochondrial concentration, thus prolonging the duration of state 3 respiration over 120 seconds. If flux reaches a constant value at state 3, then the maximum value is represented accurately by the plot of flux, and routine analysis is possible in DatLab 4.
- Export the data into DatLab 2, and apply a time correction (signal deconvolution), as described by Gnaiger 2001 RespPhysiol.
- See also ‘Notes on Time resolution’.
