Abstract
Monoclonal antibodies to human plasma fibronectin were used to study the topological arrangement of several biologically active sites on the 220,000-dalton fibronectin subunit. Plasma fibronectin was cleaved into a number of biologically active fragments by trypsin and cathepsin D. Fragments that bind gelatin and heparin bind to both gelatin and heparin were isolated by affinity chromatography. These fragments were further characterized by their ability to bind to two different monoclonal antibodies: monoclonal 2-8 and monoclonal 180-8. Using this approach, we have established the positions of two unique heparin-, a gelatin-, and two monoclonal antibody-binding sites on the fibronectin subunit.
Highlights
Monoclonal antibodies to human plasma fibronectin onto the 220,000-dalton fibronectin subunit
Fibronectin Isolation-Fibronectin was isolated from whole human plasma by affinity chromatography on gelatin-agarose by the method of Engvall and Ruoslahti [11].Affinity purified fibronectin was chromatographed on a Whatman DE52 ion exchange colbonds near the COOH terminus [7]
Monoclonal Antibody-producingClones-Fusion of splenocytes from BALB/C mice immunized with human plasma fibronectin with NS-1 mouse myeloma cells resulted in the establishment of stable,anti-fibronectin-producinghybridoma clones
Summary
Proteolytic digests were dialyzed against PBS it possible to isolate and purify fragments which bind gelatin supplemented with 1mM EDTA, pH 7.4, forchromatographic analysis [10, 14, 17,23], heparin [16,17,18], fibrin [12], and staphylococci or diluted in sample buffer for analysis by polyacrylamide gel electro-. Fragments that bound to heparin-Sepharose were eluted by increasing the concentration of sodium chloride in the PBS, 1 mM EDTA, pH 7.4, buffer to 1 M. After addition of a 10fold molar excess of pepstatin A over enzyme, the digest was chromatographed on heparin-Sepharose and the fragments in the void volume were chromatographed on gelatin-agarose. The apparent molecular weight (M.) was estimated by using plasma fibronectin (220K), phosphorylase b (93K). bovine serum albumin (68K),ovalbumin (43K),and soybean trypsin inhibitor (21K) as standards
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