Purpose/Principle

Using the current method, four different complexes are assayed in fibroblasts: complex I, II, IV, and V . After isolation of the mitochondrial membranes containing the electron transport complexes, the complexes are separated on a non-denaturing blue native PAGE gel. This separates the complexes from each other and from other interfering enzyme activities that typically complicate the interpretation of classic enzyme activity analysis in whole homogenates. The gel is then cut into four or five parts if assaying complex III activity, and the activity of each complex is analyzed by an in-gel enzyme activity with a color end product. The activity is analyzed by comparison to the same assay of control tissue run on the same gel prepared in the same batch. The assay is qualitative.