Biomedical Engineering Reference
In-Depth Information
Heating up the solution signifi cantly increases the solubility of
digitonin.
5. Alternatively, 2 mM NADH plus 2 mM ATP might be added.
Wild type mitochondria usually contain suffi cient internal sub-
strates, i.e., the addition of substrates will not further increase
the fl uorescence quenching.
6. Fluorescence intensity will not reach starting (maximal) value
again, as you will always have unspecifi c fl uorescence quench-
ing to a certain extent.
7. For preparation of a master mix, it is useful to always use
5-10 % in excess of the volumes needed.
8. To maintain the full import-competence, do not vortex mito-
chondria at all before performing the import reaction as this
might result in damages of the membranes.
9. Other detergents such as Dodecylmaltoside or Triton X-100
may also be suitable.
10. Prepare the BN gel always in advance and keep it at 4 °C until
it is loaded.
11. Addition of sodium azide to a fi nal concentration of 0.02 %
(from a 100× stock) and storage at −20 °C is useful to preserve
the antibody solution for subsequent experiments. Handle
sodium azide with care as it is highly toxic.
References
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Mitochondrial protein import: from transport
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