In-gel ligand blotting with 125I-heparin for detection of heparin-binding growth factors.
125I-labeled heparin was used to detect basic fibroblast growth factor (bFGF) in crude tumor cell extracts after separation by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. 125I-labeled heparin bound avidly to native recombinant bFGF immobilized on nitrocellulose and eluted with 1.5-2.0 M NaCl. However, Western transfer of bFGF to nitrocellulose after SDS-polyacrylamide gel electrophoresis destroyed heparin-binding ability. In contrast, bFGF was detected by incubation of the polyacrylamide gels directly with 125I-labeled heparin in a gel overly technique. Heparin affinity and NaCl elution pattern from bFGF in gel were similar to those observed for native bFGF spotted on nitrocellulose. This procedure permitted detection of bFGF in crude extracts of a human astrocytoma cell line. In view of the rapid growth of the heparin-binding fibroblast growth factor gene family, this technique should prove useful for the rapid and sensitive detection of other heparin-binding growth factors.
Murphy
P R
PR
Department of Physiology, University of Manitoba, Winnipeg, Canada.
Katsumata
N
N
Sato
Y
Y
Too
C K
CK
Friesen
H G
HG
D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D005346:Fibroblast Growth Factors / Q000302:isolation & purification / Q000378:metabolism; D006133:Growth Substances / Q000032:analysis*; D006493:Heparin / Q000032:analysis* / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D011994:Recombinant Proteins; D014407:Tumor Cells, Cultured
D006133:Growth Substances; D008024:Ligands; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; D005346:Fibroblast Growth Factors; D006493:Heparin