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Trouble with subcellular fractionation

Trouble with subcellular fractionation - Protein Science

Trouble with subcellular fractionation - Post Questions and Discuss Protein Structure and Function, Protein Localization etc.. Please do not post Proteomic questions here, there is a Proteomic Forum below.


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Old 04-26-2008, 04:30 PM
Pipette Filler
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Default Trouble with 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.
Thanks in advance
Tiffy
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