I was wondering what people
>thought of the relative merits of
>conjugating FITC to the antibody myself vs.
>buying a generic anti IgG-FITC premade conjugate and using a sandwich
>type technique.
>
>What are the pro's and cons of the various ideas?
>
I think the choice will be determined by the experimental question being
asked.
Directly conjugated antibodies are useful in multicolor labelling, are
easier [one step] and good if low cell number might be a problem [less
washes]. Remember, depending on the antigenic densityl of the marker in
question, you will probalby also need to conjugate an isotype Ab to assess
background labelling.
Indirect labelling works well for a single color assay [can be used in
multicolor but not as simple]. It is probably easier to control for
backgoround with an indirect assay [use an isotype cont and the same 2ndary
you use for your test sample]. Also, you have a choice of colors for the
GAM reagent and you may want to use something "brighter" than FITC,
depending on the resolution between your positives and negatives. All else
being equal, an indirect labelling procedure will tend to amplify your
signal as well.
Good luck,
Tom
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Thomas W. Mc Closkey, Ph. D.
Director, Flow Cytometry
North Shore University Hospital
Biomedical Research Center
350 Community Drive
Manhasset, Long Island, New York 11030
ph: 516-562-4844 [office]; 516-562-1135/4641 [lab]
3/28/97 12:52:08 PM
E-mail: thomasm@nshs.edu
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