Biology Reference
In-Depth Information
19. If the brake is engaged during deceleration the gradient frac-
tionation achieved during centrifugation will be lost due to
sample mixing.
20. We confi rm protein concentration at this stage prior to further
analysis using the Bradford protein assay [
53
].
21. The performance of oxygen electrodes deteriorates over time
due to electrochemical deposition of chloride and oxide salts
on the silver anode. It is therefore necessary to periodically
clean the anode using aluminum oxide polishing paste.
22. It is important not to add too much mitochondrial suspension;
otherwise the rate of oxygen consumption will exceed the
response time of the oxygen electrode. It is therefore advisable
to test several different concentrations of mitochondrial sus-
pension and choose one in which the oxygen consumption rate
in the presence of substrate gives an appropriate rate.
23. Aconitase activity can be determined through the measure-
ment of isocitrate production from citrate. Isocitrate produc-
tion rate is measured by activity of an isocitrate- and
NADP-dependent enzyme. Aconitase contains a Fe-S center
that is readily damage by H
2
O
2
-inhibiting activity of the pro-
tein, and the protein itself has been shown to be degraded
during prolonged oxidative stress.
24. Full solubilization should occur in around 20 min using a
pipette and vortex mixer to disrupt pellet. When using whole-
tissue extracts, the resuspension step can be notoriously prob-
lematic, but typically mitochondria proteins resuspend
relatively easily.
25. The time the CyDyes are exposed to light should be mini-
mized, as the dyes are liable to bleaching.
26. Some proteins are insoluble in this buffer, so we have found
that a centrifugation step prior to IEF removes insoluble pro-
teins that cause vertical streaking on the fi nal image.
27. Low-fl uorescent glass plates are available from GE Healthcare
Life Sciences.
28. We use an orbital rocker set to ~150 rpm, ensuring that the
“gel side” of the strip is facing upwards, to prevent bound
proteins from rubbing against the base of the container.
29. We use a 0.75 mm thick spacer to press the strip against the top
of the gel, and take care to minimize the number of air bubbles
between the strip and the gel. It is important to heat the over-
lay solution to around 50 °C to melt the agarose, but do not
overheat, as this might strip the proteins off the IEF strip.
30. We use the Ettan Dalt 6 system, and in our hands, a typical gel
requires 270 mA h of electrophoresis. For convenience, we
usually run this step overnight (i.e., 15 mA per gel for 18 h).
