..for knowledge, please. This is my week for funky questions--I'm thinking of changing our name to the "Bead Analysis Facility". I'm hoping (counting on) someone knowing more about quenching than I do. I have a user who's working out a DNA binding competition assay, and they're looking at detection methods. They were thinking about incubating oligo-labelled beads with a fluorescent complimentary strand, and then incubating with the analyte. Competition would knock off the label and reduce fluorescence. Now they're thinking about starting with beads with fluorescently labelled oligos, incubating them with complimentary probes conjugated to quenching molecules (blocking the bead fluorescence), and then doing the analyte incubation. Successful competition would then knock off the quenchers and increase fluorescence. Any opinions ? I'm aware that Trypan blue quenches FITC, but is this a binary (on/off) phenomenon? Does the degree of quenching vary? Can trypan blue be stably conjugated to oligos? Are there other molecules out there to consider, or any source of info that someone could recomend? My gut impression is that it won't make much of a difference--it seems to me that our signal/noise ratio is going to depend on how many FITC molecules are knocked off (protocol a) or exposed (protocol b). I can't see why one approach would be better than the other, and if the quenching response varies at all it seems like it just introduces one more source of variation. I realize this is a bit vague, but any insights you can offer would be gratefully accepted. And thanks for the magnetic bead feedback to date, Steve +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ Steve G. Hilliard (706) 542-9474 University of Georgia Cell Analysis Facility flowman@uga.edu http://floweb.cb.uga.edu/
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