Need help on cell fractioning

Martin Offterdinger martin.offterdinger at akh-wien.ac.at
Thu Aug 17 04:04:22 EST 2000


Stephen Snowdy <snowds at med.unc.edu> schrieb in im Newsbeitrag:
399AB63F.7F40ABF at med.unc.edu...
> I'm fractioning cells (HeLa) into cyto vs nuclear fractions by hypotonic
> treatment and dounce homogenization.  What I need is positive control
> proteins for the nuclear and cyto fractions.  I was thinking histone H1
> for the nuclear, but I'm not sure whether it comes out in the soluble
> nuclear fraction or the insoluble.  Any ideas for the soluble cyto
> fraction?
> --
> Stephen Snowdy
Stephen Snowdy <snowds at med.unc.edu> schrieb in im Newsbeitrag:
<399AB63F.7F40ABF at med.unc.edu>...
> I'm fractioning cells (HeLa) into cyto vs nuclear fractions by hypotonic
> treatment and dounce homogenization.  What I need is positive control
> proteins for the nuclear and cyto fractions.  I was thinking histone H1
> for the nuclear, but I'm not sure whether it comes out in the soluble
> nuclear fraction or the insoluble.  Any ideas for the soluble cyto
> fraction?
> --
> Stephen Snowdy

Histone H1 is suitable; I used it for the same purpose and it worked (using
the Neomarkers antibody)
Cytoplasm: Do not use actin; it is partially polymerized and insoluble and
comes down with the nuclei; if you high salt treat them afterwards you
solubilize the actin filaments---> this causes a very high apparent stain of
the nuclear fraction.
You can use GAPDH; there is one antibody from a company called Advanced
Immunochemical (no affiliation!). I used it and it worked very well.
Martin







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