Hi Michelle...
Although I'm not completely sure what the "5-EX" stands for, I'm assuming
that it may be the single-isomer form of carboxyfluorescein (the "5") and
that the "EX" stands for a spacer arm (the 5 might alternately stand for
the length of the spacer arm). In our hands, succinimidyl ester
conjugations are more reliable and consistent than isothiocyanate
couplings, so we prefer the SE reactive forms for doing all our labelings
(fluorescein, rhodamine, biotin, etc.). There are some cases where
antibodies label well by one method and poorly by another, though (or one
labeling method blocks the antigen binding site and the other doesn't), so
you should keep this in mind if you switch over to SE chemistry.
For the "5" part, for flow we have found that single or mixed isomer
preparations of carboxyfluorescein don't make any great difference. And we
use reactive forms with spacer arms (the "X" part) whenever possible, since
they position your fluorochrome away from the protein you are
labeling. Size difference with FITC should be minimal and should not
affect intracellular labeling.
You might also want to consider Molecular Probes Alexa Fluor 488 - we find
it to be more resistant to photobleaching than fluorescein, and is
spectrally interchangeable. MPI sells a conjugation kit and (I think) the
SE form alone, too.
Enjoy,
Bill Telford
NCI-NIH
Disclaimer: I have no financial stake in MPI !!!.
At 05:54 AM 9/4/2002 -0400, michelle connole wrote:
>Does anyone have any experience/opinion as to whether
>Fluorescein-5-EX succinimidyl ester would be a preferred choice over
>Fluorescein isothiocyanate (FITC) for conjugating a mouse monoclonal
>IgG Ab (for SIV gag antigen detection). Our concern is that since
>this antibody will be used for intracellular staining that there may
>be issues with the size difference between the two which may negate
>any advantages that the Fluorescein-EX would have over FITC.
>Your input is very much appreciated.
>
>Michelle Connole
>New England Primate Research Center
>Harvard University
>1 Pine Hill Drive
>Southborough,MA
>01772
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