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> Let me suggest that you consider a completely different approach to
> your assay. Rather than attempting to detect the beta-galactosidase protein
> directly by immunofluorescence, you can take advantage of beta-galactosidase's
> enzymatic properties. A compound called (if I remember correctly) fluorescein
> digalactoside (FDG) is introduced to the transfected cells. The cells that
> possess beta-galactosidase activity convert FDG into something like carboxy-
> fluorescein, which is both fluorescent and membrane-impermeant.
> [underline mine...RA]
> This flow cytometry assay is exactly analogous to the standard
> colorimetric assay for
> beta-galactosidase activity... which you've already done in order to deter-
> mine that your cells really have been transfected, right?
However, be very careful here. We have seen non-transfected cells pick
up fluorescein from the transfectants when the non-transfected cells
were added to the transfected cells after FACS-gal staining. So good
controls are in order to be sure that, in fact, the positives are really
beta-galactosidase positive. While the enzymatic prodict is stated to
be trapped in the cell one should ensure this for ones own cells...
***************************************************************
Richard B. Alexander rich@alw.nih.gov
National Cancer Institute Voice (301) 496-3098
Building 10, Room 2B56 Facsimile (301) 402-0922
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