Jeannine, You'll want to relate the isotype to its companion(s) . . . so, whatever concentration you choose for the specific antibodies, compare that to an equal concentration of the matching isotype. This is best related in equivalent amounts of protein, not simply a dilution factor, so you do need to assay for content. It's also important (many may say most important) to compare fluorochrome/protein ratio of controls and specific antibodies. Differences in conjugation efficiency will dramatically effect the eventual fluorescence, and will also effect "noise" detected with an isotype. This has been one of the many arguments against the use of isotypes in general . . . since the F/P ratio is often different, using these as controls for establishing +/- cutoffs may be misleading. Personally, I believe isotypes are useful in most cases in the absence of better negatives, and if one accounts for protein content and F/P differences. I hope this doesn't prompt a new "isotype vs. no isotype" debate . . . MAK. -- Mark A. KuKuruga, Managing Director University of Michigan Flow Core 7416 CCGC 0946 (734) 647-3216, fax (734) 936-7376 kukuru@umich.edu
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