Purification of human plasma fibronectin using immobilized gelatin and Arg affinity chromatography.
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.
Speziale
Pietro
P
Department of Biochemistry, Viale Taramelli 3/b, 27100 Pavia, Italy. pspeziale@unipv.it
Visai
Livia
L
Rindi
Simonetta
S
Di Poto
Antonella
A
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
D005353:Fibronectins; D005780:Gelatin; D001120:Arginine