Separation of three fractions from mammalian cell lysates

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salmon
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Separation of three fractions from mammalian cell lysates

Post by salmon » Wed Sep 08, 2010 4:51 am

Dear friends and experts,

I am trying to obtain 3 fractions (nuclear, cytoplasmic and membrane) after mammalian cells (Caco-2 cells) are lysed using the NP-40-based lysis buffer (see below). Here I have two questions:

1. Will 500 × g for 10 min at 4ºC be sufficient to obtain nuclear fraction? Any better way to do it?
2. Under what condition can I obtain the membrane fraction?

Thank you!

Lysis buffer:
------------------------------------------------------------
50 mM Tris pH 8.0
0.4% NP-40
125 mM NaCl
1 mM Na3VO4
25 mM NaF
1 mM EDTA
Complete protease inhibitor (Roche)

:D

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