Help! Panning

Brett brett at BORCIM.WUSTL.EDU
Tue Aug 8 11:47:17 EST 1995


>In article <9508080226.AA12954 at gipac.shinshu-u.ac.jp>,
>tnakane at GIPAC.SHINSHU-U.AC.JP (tokio nakane) wrote:
>
>> Hello,
>> We are currently trying to clone a G protein-coupled receptor by panning
>method.
>> We constructed a rat brain cDNA library (pME18S vector; SR-alpha promoter)
>> and transfected the library to COS-7 cells by DEAE dextran method.  After
>> 72 hrs, we could not detach cells from dishes by the treatment with PBS and
>> 5 mM EDTA at 37 C for 30 min.  The untransfected cells were easily detached
>> by the treatment.  Any advice would be much appreciated.
>> 
>> Tokio    
>
>Dear Tokio,
>
>You might try 293 cells (human embryonic kidney).  They are generally
>easier to detach from plates.
>
>Cliff Beall

Cliff's suggestion is well-taken, but I don't think it's what Tokio should do.
The higher he can get expression levels, the more efficient his panning will
be. The SR-alpha promoter contains most of the SV40 origin, and so should give
exteremely high levels in COS-7 cells, which are SV40 T(+). 293 cells on the
other hand, were transformed by Ad5. I would instead plate the cells out on
100mm sterile petris, *not* tissue culture treated, shortly after
transfection. Your cells may adhere, but are unlikely to spread. Detachment
should then be
no problem. If this strategy proves unfeasible, break out you scraper, Tokio.
Good Luck!



Brett Lindenbach
    
Program in Immunology                              
Washington University - St Louis                  
brett at borcim.wustl.edu                             






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