Nuclear Staining + Protein Isolation??

Wolfgang Schechinger hubahopp at gmx.de
Mon Jul 26 08:46:06 EST 2004


Dear Samantha,

for nuclear staining, simply use DAPI or Hoechst H33258 (bis-benzimidazol).
Detergents might kill your GFP fluorescence. 

for nuclear proteinisolation, first prepare nuceli (eg with triton or NP40
in buffer, your nuclei will remain on the dish but cell membranes will be
disrupted and cytosol will be washed away except the cytoskeleton. Then
collect nuceli and extract proteins, Maybe it's necessary to add DNAse to
reduce viscosity caused by nuclear lysis.

Best regards,

Wolfgang

At 23:05 24.07.2004 +0800, you wrote:
>Hi,
>
>i had transfected wells with pEGFP-my gene (which is localised in the
>nucleus)
>then..i wanted to stain the nucleus to check the transfection efficiency
>i used3.7% paraformaldehyde to fix cells and 0.05% triton-x 100 to
>permealise the membrane (All in 1X PBS and the cells were wahsed with 1X
>PBS)
>after the staining, i wanted to isolate the proteins
>so i tried to use the normal protocol to prep the proteins (that protocol is
>fine for other proteins which had not been stained: just used trysin to
>trysinize the cells and lysis buffer we commonly use)
>but i could not harvest the proteins
>
>** I want to ask it is fine to stain the cells and then harves the proteins?
>(I ve tried to find the solutions on the web, but I couldnt)
>
>Thank you very much.
>
>Samantha
>
>
>
Wolfgang Schechinger


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