Nuclear Staining + Protein Isolation??

Kyle Legate legatek at hotmail.com
Sun Jul 25 03:22:43 EST 2004


Samantha 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)
>
If you're trying to check for transformation efficiency using a GFP-labelled
protein, why are you fixing and permeabilizing? Just grow your cells on a
cover clip and look under a fluorescence microscope. As soon as you fix your
cells you ruin any opportunity to obtain a protein lysate since fixing works
by crosslinking protein.





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