Lasting effect of EtOH-soluble inhibitors
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Revision as of 13:44, 7 September 2010 by Sumbalova Zuzana (talk | contribs)
Problem
Lasting effect of inhibitors (i.e. rotenone, antimycin A), as observed by persistent low fluxes, when working with permeabilized fibers.
Cleaning the Chamber
Clean the chamber after an experiment involving lipid-soluble inhibitors (such as oligomycin, rotenone, or antimycin A):
- The chamber must be cleaned rigorously with ethanol, since such inhibitor(s) are difficult to be washed out from the chamber and may inhibit mitochondrial respiration in subsequent experiments.
- Siphon off the cell/mitochondrial suspension at the end of the experiment and rinse the chamber with distilled water three times, by filling the chamber up to the rim.
- Also rinse the surface and capillary of the stopper with distilled water properly.
- Fill the water-cleaned chamber with 70 % ethanol and replace the stopper making sure that the ethanol fills up the receptacle, and leave for 5 min.
- Remove the stopper and siphon off the ethanol to empty the chamber. Repeat these cleaning steps with 70 % ethanol three times.
- Then fill the chamber with absolute ethanol (99.6 %) and insert the stopper making sure that the ethanol fills up the receptacle. Place the perspex cover on top of the stopper and leave for 15-20 min.
- At the end of this rigorous cleaning procedure remove the stopper and place it inverted (with the receptacle at the bottom) on a clean paper towel.
- Siphon off the ethanol from the chamber and rinse it with distilled water three times.
- Rinse the stopper by holding it at the receptacle, not at the shaft that fits into the chamber to avoid contamination.
