Transfection of proteins: HOW?

Nikolai Chitaev nikolaic at VISAR.WUSTL.EDU
Fri Mar 8 14:35:49 EST 1996


Stefan wrote:
> Stefan Kahlert (UZS13B at ibm.rhrz.uni-bonn.de)
> Mon, 04 Mar 96 10:41:13 MEZ
> I want to block proteins in cells with specific antibodies,
> and I'm looking for possible methods to get this antibodies
> into these cells.
> Problems:
> 
> - microinjection is not possible because I work with suspension cells
> (hard to catch and even harder to find the injected ones to check
> the result)
> 
> - electroporation is *not* the way to (please don't ask why. The
> story is to long to be told here I think).
> 
> What other methods are possible to get antibodies or other proteins
> into suspension cells?
> 
> Thanks in advance

Besides lipofection and electroporation, I`d
suggest to fix the problem, by fixing cells to the solid support and then
use convinuent microinjection as usual.
Cells coud be fixed to the bottom of the dish using antibodies agains surface
receptors and an addition, perhaps, of paraformaldehyde or other crosslinking 
agents.
As a first step, coat the dish with antibodies agains some of the adhesion 
receptors. Put the cell`s suspension in there in PBS, wait untill cells will be
attached to the surface of the dish. At this point you are either done or half
as done depending how well your Ab works. If you feel that Ab works poor then try
to fix Ab/Ag interaction by adding some of the crosslinking reagents (see PIERCE 
catalogue). Finaly you should get your cells attached to the surface of the dish
strong enough to performe microinjection of your protein into the cells. You even
don`t need to detach cells later after the microinjection.
Another possibility is to use convinient crosslinking reagent(s) to attach the 
cells directly to the dish. Good luck.

Nikolai
http://128.252.119.253






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