Organelle isolation: functional mitochondria from mouse liver, muscle and cultured filroblasts
Mitochondria participate in key metabolic reactions of the cell and regulate crucial signaling pathways including apoptosis. Although several approaches are available to study mitochondrial function in situ are available, investigating functional mitochondria that have been isolated from different tissues and from cultured cells offers still more unmatched advantages. This protocol illustrates a step-by-step procedure to obtain functional mitochondria with high yield from cells grown in culture, liver and muscle. The isolation procedures described here require 1–2 hours, depending on the source of the organelles. The polarographic analysis can be completed in 1 hour.
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NOTE FROM NATURE PROTOCOLS EDITORS
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Posted by: Dorothy Clyde | April 8, 2009 11:21 AM
There is a mistake in the Reagent Setup. The preparation for Experimental buffer for cell and mouse-liver mitochondria should read: mix 12.5 ml of 1 M KCl, 1 ml of 1 M Tris/MOPS, 10 muL(read microliters) of 0.1 M EGTA/Tris and 100 mul of Pi. Adjust pH to 7.4. Bring the volume to 100 ml with distilled water.
In this way the final concentration of EGTA will be 10 muM, as it should be.
Posted by: Bronwen Dekker | October 6, 2009 04:27 PM