Subcellular fractionation

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Tiffy's picture
Subcellular fractionation

Hello, actually I have found a good protocol for fractionate cytoplasm and nuclei of cells. In particular I turn my attention to the nuclei. But lately I always got cytoplasmic contamination in the nuclei fraction. Method briefly described:
lysis with a buffer (0.1%TX100), dounce 10 strokes, 500g 6 min, supernatant discard, pellet resuspends in buffer and layers over sucrose buffer (45%), 1600g 30 min, pellet wash with surcose buffer (10% + MgCl2),dounce 7 strokes, 500 g, 6 min, Pellet takes up in buffer. I have prepared all buffers new. No success.
I take equal amounts of sucrose buffer and resuspended nuclei solution at the sucrose gradient. Shall I repeat the gradient or shall take more sucrose buffer? Please help me. Tx