Re: enhanced chemiluminescence vs. flow cytometry
Ray Hicks (rh208@cus.cam.ac.uk)
Wed, 14 Feb 1996 18:36:08 +0000 (GMT)
Hi Alan,
What is the antigen? Is it possible that it leaches from permeablised cells,
and is not available for staining in flow cytometry? Is it possible that it is
secreted and again isn't available for intracellular staining but is picked
up mainly in the supernatant by ECL?
On the apples and oranges front, background might also be an issue; if the
antibody is cross reactive you might see a large number of dim bands on a gel
and one intense one, on a flow cytometer the "intense" protein's signal
may not be all that remarkable compared to the sum of the minor proteins'
signals.
Ray
On Tue, 13 Feb 1996, Alan Salkind wrote:
> We observe relatively small changes (approximately 10%) in the mean
> fluorescence intensity of an intracellular antigen by flow cytometry.
> However, when we analyze cell lysates by immunoblotting and ECL the signal
> is significantly increased. Is this a problem of comparing apples with
> oranges? Or is ECL much more sensitive for detecting small changes in
> antigen levels?
>
>
> Thanks in advance for your comments and ideas.
>
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