title: Purification of human plasma fibronectin using immobilized gelatin and Arg affinity chromatography.
abstract: This protocol describes a method for purification of fibronectin (Fn) from human plasma based on a combination of gel filtration and affinity chromatography steps. Clarified plasma is first loaded onto a Sepharose CL-4B column and unbound material is sequentially purified on columns containing covalently coupled gelatin and Arg. The elution conditions are optimized to obtain a homogeneous preparation of Fn on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Although the Fn yield is expected to be lower than that obtained using other methods, affinity adsorbents based on gelatin and Arg and gentle elution steps offer advantages including a high purity of the preparation and a correctly folded protein. The preparation can be useful for interaction studies and analysis of biological and immunological activities of Fn.
authors.affiliation: Department of Biochemistry, Viale Taramelli 3/b, 27100 Pavia, Italy. pspeziale@unipv.it
authors.forename: Pietro
authors.initials: P
authors.lastname: Speziale
authors.forename: Livia
authors.initials: L
authors.lastname: Visai
authors.forename: Simonetta
authors.initials: S
authors.lastname: Rindi
authors.forename: Antonella
authors.initials: A
authors.lastname: Di Poto
mesh_terms: D001120:Arginine / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D002847:Chromatography, Agarose / Q000379:methods; D005353:Fibronectins / Q000097:blood / Q000737:chemistry* / Q000302:isolation & purification*; D005780:Gelatin / Q000737:chemistry; D006801:Humans
chemical_list: D005353:Fibronectins; D005780:Gelatin; D001120:Arginine