LABELING OF BASIC FIBROBLAST GROWTH-FACTOR WITH DIGOXIGENIN - A NONRADIOACTIVE PROBE FOR BIOCHEMICAL AND CYTOLOGICAL APPLICATIONS

Digoxigenin, a 391-Da plane 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 c onjugate, bFGF-DIG, displayed a mitogenic activity on endothelial cell s equivalent to that of nonlabeled bFGF. Binding of the probe on the c ell surface was assessed by ELISA on cells, which allowed discriminati on between low- and high-affinity bFGF binding sites. Using a chemilum inescent system, chemical cross-linking of bFGF-DIG with FGF receptors was analyzed directly on Western blots of cell extracts with anti-dig oxigenin antibodies. The labeling pattern was identical to that report ed with iodinated bFGF, showing that bFGF-DIG bound to the same recept ors. The time course of intracellular degradation of internalized bFGF -DIG was also followed by immunodetection on Western blots: the low sp eed 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 cultu red cells, and was shown to be an interesting probe to determine bFGF endocytosis pathways by electron microscopy. bFGF-DIG appeared as a mu ltifunctional nonradioactive probe suitable for combined biochemical a nd cytological studies of bFGF. (C) 1994 Academic Press,Inc.