--- On Thu, 27 Mar 1997 00:06:24 -0500 (EST) Dave Novo <novod@muss.cis.mcmaster.ca> wrote: 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 -------------------------------------------------------- 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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