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Open Forum on Methodology

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Cell fractionation

12 July 2005

Bakary Sylla

July 11, 2005

Question on cell fractionation:

Does anyone have a good protocol (or suggestion, or explanation) for cell fractionation in order to separate nuclear from cytoplasmic fractions? We are trying to show that a protein that shows a clear nuclear localization in immunofluorescence assay can be found predominantly in the nuclear fraction. However, using the cell fractionation protocol we have, the protein shows equal distribution between cytoplasm and nucleus, whereas the control proteins (tubulin and PARP) show the expected distribution. We think that probably our protein is loosely present in the nucleus and becomes easily leaky during the process.

Thank you for your help and suggestions.

Regards,

Bakary

Protocol being used:

  • Cells are resuspended in buffer A (10mM HEPES, pH 7.9, 1,5 mM MgCl2, 10 mM KCl, 10% Gycerol, 340 mM sucrose) supplemented with protease inhibitors.
  • Add Triton X-100 to 0.1%
  • Leave on ice for 5 min
  • Spin at 1300g for 4 min at 4 degrees
  • Transfer the supernatant to a new tube (cytoplasmic fraction)
  • Wash the pellet (nuclei) with buffer A and add 1XSDS- PAGE buffer, sonicate to obtain the nuclear fraction

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