nucleus removal in Sf9 cells.

Dima Klenchin klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu
Sat Dec 27 21:41:22 EST 1997


In article <67f3pf$te$1 at agate.berkeley.edu>, lhom at nature.berkeley.edu (Louis Hom) wrote:
>In article <tyr-2-1812972104370001 at news.srv.ualberta.ca>,
>Karl Fischer <tyr-2 at bones.biochem.ualberta.ca> wrote:
>>"NP-40 will destroy the membrane"....hmmm.
>
>I second the hmmm, sort of.  I have no doubt that NP-40 will
>solubilize the nuclear membrane just as it does the plasma membrane.  But I
>don't think it will cause too many problems if all you want is to avoid the
>release of the DNA.  The nonmembranous elements of the nucleus
>(cytoskeletal, lamins?) are generally quite stable to low concentrations of
>nonionic detergents like NP-40 -- infected Sf9 nuclei especially so.  I'd
>recommend starting with an NP-40 concentration of around 0.2% and pelleting
>out the nuclei (and stuff) with a 5 minute spin in the microfuge
>(15krpm?).  No vortexing should be necessary.

NP-40 and Triton X-100 chemically are the same thing. Both at concentrations up to
1% solubilise cells leaving nuclei "intact". It's very common practice. 

- Dima





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