By Yu-Li Wang, D. Lansing Taylor and K.W. Jeon (Eds.)
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Extra info for Fluorescence Microscopy of Living Cells in Culture Part A . Fluorescent Analogs, Labeling Cells, and Basic Microscopy
J. Biol. Chem. 259,4812-4821. Kahn, C. , Baird, K. ,Jarrett, D. , andFlier, J. S. (1978). Proc. Acad. 75, 4209-4213. , andohnuma, H. (1976). Biochim. Biophys. Acfa351, 205-21 3. Keen, J. , Maxfield, F. , Hardegree, M. , and Habig, W. H. (1982). Proc. Acad. Sci. 79,2912-2916. 28 FREDERICK R. MAXFIELD Levitzki, A. (1985). In “Endocytosis” (I. Pastan and M. C. Willingham. ), pp. 45-68. Plenum, New York. Maxfield, F. R. (1985). In “Endocytosis” (I. Pastan and M. C. ), pp. 235 -257. Plenum, New York.
In many laboratories, equipment for measuring binding of radiolabeled hormones is more readily available than equipment for quantifying fluorescence in cells. The ability of the fluorescent-labeled hormone or peptide analog to compete for binding sites with a radiolabeled analog can be measured using standard techniques for determining the Kd of a radiolabeled hormone (Levitzki, 1985). Although this procedure is simpler than measurements of the fluorescence intensity, it has several limitations.
Typically, three peaks are observed. The first two are somewhat closely spaced and on occasion overlap, whereas the third is separated by at least eight column volumes. Only the first two fractions should show fluorescence corresponding to the TmRhd moiety.