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You may also be seeing a protein either partially or completely embedded
in the membrane, more accessable to the antibody when the membrane is fixed.
OR your surface protein could be shed by the cells.....
Good luck, hope this rambling helps.
Deb Berglund
Montana State University
On Mon, 7 Jul 1997, Kevin G Waddick wrote:
>
>
> Hi everybody,
> I am puzzled by a question that I hope someone can help me understand.
> We are using a human lymphocyte cell-surface protein extracellular domain
> that, through stable transfection, is being produced in insect cells.
> Although this antigen itself is well-characterized, and it seems to be a
> receptor protein, no counter-ligand has been definitely found yet.
> Here is the problem. We have biotinylated the protein and are testing
> a wide range of different cell types for the ability to specifically bind
> it. When we test unfixed cells by flow cytometry, we either get no
> staining (using either avidin-FITC or -PE) or when there seems to be some
> staining, it is merely 1-10% of the primary or cell line cells with a
> large range of intensities (starting at barely above the
> control-determined +/- level). So far so good, however, when a guy here
> did side-by-side fluorescence confocal microscopy, he used cells that
> either were or were not formaldehye-fixed prior to treatment with protein
> followed by the avidin-fluorochrome; the fixed cells of a particular type
> showed a definitely positive fraction of ~50%, while the unfixed cells had
> a positive percentage similar to the unfixed cells examined by FCM. When
> previously fixed cells were tested using FCM, a tightly-focussed,
> reasonably intense positive population of ~60% appeared, whereas in the
> unfixed sample the positivity was only 5% with the usual scattered
> intensity. The staining can be partially blocked using unbiotinylated
> ligand.
> I don't think the staining of the fixed cells is nonspecific because
> cell types that were totally negative when unfixed remain negative when
> fixed. It seems contrary to sense that if a cell has a specific receptor
> for a ligand, it only becomes highly evident using fixed cells and is
> questionable when using healthy, unfixed cells. Does anyone have
> experience with this phenomenon? What does it mean?
>
> Kevin G. Waddick, Ph.D.
> Staff Scientist
> Hughes Institute
> 2685 Patton Road
> Roseville, Minnesota 55113
> (612) 628-0598
> (612) 628-9891 Fax
>
>
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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;
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