Labeling of basic fibroblast growth factor with digoxigenin: a nonradioactive probe for biochemical and cytological applications.
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.
Gleizes
P E
PE
Laboratoire de Biologie Moléculaire Eucaryote (UPR 9006-CNRS), Toulouse, France.
Noaillac-Depeyre
J
J
Gas
N
N
D000818:Animals; D001013:Aorta, Thoracic; D015153:Blotting, Western / Q000379:methods; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002478:Cells, Cultured; D006224:Cricetinae; D004076:Digoxigenin; D004705:Endocytosis; D004730:Endothelium, Vascular / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D016222:Fibroblast Growth Factor 2 / Q000032:analysis* / Q000378:metabolism / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D008854:Microscopy, Electron / Q000379:methods; D017468:Receptors, Fibroblast Growth Factor / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism / Q000494:pharmacology; D012680:Sensitivity and Specificity
D007202:Indicators and Reagents; D017468:Receptors, Fibroblast Growth Factor; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; D004076:Digoxigenin