Hi Kevin,
I can't help you to understand, but finding the answers to some of these
questions might:
Did the confocal allow you to discern the localisation of the staining? If
it was on the surface it could be that you've opened up a "hidden epitope"
by fixing. Otherwise it could be that you are seeing an internal store or
nascent ligand - was it localised to ER or golgi etc? Is it posible to
blot a gel of cellular protein with your probe to see how many protein
species are involved?
Formaldehyde isn't great for permeablising cells, what happens if you add a
detergent to ensure that all of your cells are permeablised; do you still
get only 50-60%? What happens if you don't fix them, eg if you cytospin
them for microscopy, does the binding still occur? Have you included
avidin controls; there are reports that streptavidin may specifically bind
certain amino acid sequences, could this be why you only get partial
blocking with cold ligand?
good luck
Ray
At 10:25 am -0500 7/7/97, 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
Ray Hicks
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