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.
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