NP40 lysis of adherent cells
stayve-and-irayne at worldnet.att.net
Mon Nov 29 13:04:00 EST 1999
Stephen Snowdy <snowds at med.unc.edu> wrote in message
news:38418A31.3436C390 at med.unc.edu...
> Does 1% NP40 lysis release nuclear proteins, or does the nucleus remain
> intact and come down with cell debris?
Unfortunately, it depends on the protein. Some stay put some leave. Lysing
in NP40 releases many of the cell contents and a spin in the microfuge will
give you a detergent extractable sup and insoluble pellet. Microscopic
observation will show you the pellet consists of nuclei, cytoskeletal
structures, and some indescribable material. The supernatant will appear
free of this matter, but don't be fooled. We study a transcription factor
that can be "chased" into the nucleus with appropriate stimulation of the
cells. Immunofluorescence shows a high percentage of the protein in the
nucleus post-stimulation (maybe 80%), but the factor is soluble when cells
are lysed for 30 minutes in 1%NP40, 50 mM Tris pH 8.0, 150 mM NaCl, 5 mM
EDTA. (This as revealed by immunoblots.) Thus, I would suggest trying the
NP40 lysis as well as a hypotonic followed by dounce or syringe for starters
and compare to see what works best for your needs.
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