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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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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web