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To obtain optimum conditions, we varied the ratios of the immunoreagents and the antigen (BrdU or Tg). In particular, we used an excess of fluorescently labeled secondary antibody to complex with all available primary antibody; similarly, we maintained an excess of both secondary and primary antibodies over the amount of antigen. Thus, the complex of antigen with the primary antibody was completely labeled with the secondary antibody and was detected by laser-induced fluorescence. This important consideration has been demonstrated in all electropherograms, which always show the presence of peak 1 (excess unbound secondary antibody) and peak 2 (complex of primary and secondary antibodies). Peak 4, which is also present in all electropherograms, arises from the free fluorophore TMR, an impurity present in the secondary antibody reagent. We used peak 4 as an internal standard to correct for changes of instrument sensitivity. Because the primary antibody is saturated with the secondary antibody and because the fluorescence from the same secondary antibody is measured, the relative fluorescence intensity from 1 mol of Tg should be equal to that from 1 mol of BrdU. Thus, we quantified Tg from measured fluorescence intensities by using calibration against BrdU DNA standards.
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We thank N. Dovichi, S. Hrudey, and T. Lindahl for reviewing the manuscript and M. Ma, L. Ye, W. Ansbacher, and S. Kuny for technical assistance. Supported by grants from the Natural Sciences and Engineering Research Council of Canada, the National Cancer Institute of Canada, the Alberta Heritage Foundation for Medical Research through the Eco-Research Chair, the Alberta Cancer Board, and NIH (CA40453 and CA62059).
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