Detection and characterization of heparin-binding proteins with a gel overlay procedure.
The binding of 125I-labeled derivatives of heparin has been used by several investigators to identify heparin-binding fragments of different heparin-binding proteins. In this report we utilize the procedure described by J.W. Smith and D.J. Knauer (1987, Anal. Biochem. 160, 105-114) to produce 125I-fluorescein-heparin. Using this derivative, we compare the use of gel overlay procedures with "Western blot" procedures for the detection of heparin-binding proteins following polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. We show that the gel overlay procedure is a relatively simple and sensitive method for visualizing heparin-binding proteins. In addition, we use the procedure to characterize the heparin-binding properties of heparin-binding growth factor 1 (acidic fibroblast growth factor) with synthetic peptide competitors and site-directed mutants of the growth factor.
Jerome H. Holland Laboratory for Biomedical Research, Laboratory of Molecular Biology, American Red Cross, Rockville, Maryland 20850.
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Mehlman
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Burgess
D015153:Blotting, Western; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel; D016220:Fibroblast Growth Factor 1 / Q000032:analysis*; D016222:Fibroblast Growth Factor 2 / Q000032:analysis*; D005452:Fluoresceins; D006493:Heparin / Q000032:analysis*; D006801:Humans; D007457:Iodine Radioisotopes; D007553:Isotope Labeling; D011485:Protein Binding; D011994:Recombinant Proteins
D005452:Fluoresceins; D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; D016220:Fibroblast Growth Factor 1; D003101:Collodion; D006493:Heparin