Labeling of basic fibroblast growth factor with digoxigenin: a nonradioactive probe for biochemical and cytological...

Digoxigenin, a 391-Da plant sterol, was conjugated to recombinant bFGF with the aim of detecting it with high specificity and sensitivity in cultured eukaryotic cells using antibodies against digoxigenin. The conjugate, bFGF-DIG, displayed a mitogenic activity on endothelial cells equivalent to that of nonlabeled bFGF. Binding of the probe on the cell surface was assessed by ELISA on cells, which allowed discrimination between low- and high-affinity bFGF binding sites. Using a chemiluminescent system, chemical cross-linking of bFGF-DIG with FGF receptors was analyzed directly on Western blots of cell extracts with anti-digoxigenin antibodies. The labeling pattern was identical to that reported with iodinated bFGF, showing that bFGF-DIG bound to the same receptors. The time course of intracellular degradation of internalized bFGF-DIG was also followed by immunodetection on Western blots: the low speed of the catabolic process and the size of the degradation products were comparable to those previously described with iodinated bFGF. In parallel, bFGF-DIG was readily detected by immunofluorescence in cultured cells, and was shown to be an interesting probe to determine bFGF endocytosis pathways by electron microscopy. bFGF-DIG appeared as a multifunctional nonradioactive probe suitable for combined biochemical and cytological studies of bFGF.