Gentle lysis of cells in sucrose-containing Mito-Buffer |
Characteristics of this procedure: Lysis in the sucrose-containing Buffer ("Mito-Buffer") is supposed to prevent accidential disrupture of the mitochondria to prevent the leakage of mitochondrial proteins (such as cytochrome c) into the cytosol. Lysis is achieved by using a pestle homogenizer; the number of strokes with the pestle to lyse the cells but leave the mitochondria intact, depends on the cell line. For PC3, DU145, JCA1 and ALVA31 up to 50 strokes appear to be reasonable, whereas mitochondria from LNCaP appear to be more fragile and only up to 30 strokes should be applied. If different compartments are going to be isolated, the cell lysate is centrifuged at different speeds to obtain successively nuclei, mitochondria and cytosol. The method was basically described by Yang et al., 1997, Science, 275: 1129-1132. |
PROCEDURE
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MATERIAL |
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Mito-Buffer: |
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COMPOSITION: |
RECIPE for 5 ml: |
20 mM HEPES (pH 7.5) |
2 ml of 50 mM HEPES, pH 7.5 incl. |
TNC-Buffer: |
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COMPOSITION: |
RECIPE for 5 ml: |
10 mM Tris-acetate (pH 8.0) |
1 ml of 50 mM Tris-acetate, pH 8.0 |