b-galactosidase assay after transfection

Robert A. Anders ander at fermat.mayo.edu
Sun Apr 17 09:57:26 EST 1994


In article <199404141123.UAA14023 at inetnif.niftyserve.or.jp>,
HGF02633 at niftyserve.or.jp (=?ISO-2022-JP?B?GyRAQFBETT1TPCMbKEo=?=) wrote:
> 
> Does anybody know sesitive assay system to detect beta-galactosidase 
> activity in cell extracts after DNA transfection (lipofectin method). 
> We transfected pCH110 (beta-galactosidase expression vector) into PC12 
> cells. However, the transfection efficiency was very low. We could not 
> detect the enzyme activity with o-nitrophenol-b-D-galactopyranoside 
> (ONPG) as a substrate, though the CAT activity was detected in the same 
> expeirment. How can we overcome this problem. please teach me.
> 
> Sincerely yours,
> 

 I can not answer your question on the sensitivity of the
beta-galactosidase assay more than to say that it is not all that
sensitive.  However, I've found luciferase to be a much better enzyme
system than beta-gal.  Luciferase is more sensitive, easier to assay and
provides better quatitation.  Also, the luciferase system is linear over a
wide range (at least 6 orders of magnitude).  Really for all the same
reasons it is better than the CAT system.  Other thoughts on your problem. 
1) Vary the total amount of DNA from 0 to 15ug.  2) Different transfection
system such as electroporation, DEAE-Dextran or CaPO4 precip (although I
suspect your lipofectin method is better than the latter 2, some cells work
better with certain methods!?).  

I hope this helps.
RAA     



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