Plasma fibronectin: three steps to purification and stability.
Large amounts of soluble fibronectin were easily purified from cryoprecipitated or fresh citrated human blood plasma by a three-step combination of gelatin and heparin-cellufine affinity chromatography. The elution conditions were optimized to obtain a homogeneous fraction on SDS-PAGE and Western blot under reducing condition. No proteolytic activities were detected by zymography at acidic or neutral pH. Furthermore, the fibronectin preparation was stable over time up to 456 h at 37 degrees C in the presence of calcium, zinc, or mercury. This preparation of very stable fibronectin, called highly purified fibronectin (hpFN), gave a yield of 7.00 +/- 0.77 mg of fibronectin per gram of cryoprecipitated plasma and 0.16 mg of fibronectin per milliliter of fresh citrated, giving a yield of 32 to 53% (from presumed fibronectin concentration). This preparation may be useful for cellular tests and interaction analysis.
Poulouin
L
L
Equipe de Recherche sur les Relations Matrice-Extracellulaire Cellules (ERRMECe), Université de Cergy-Pontoise, 2 avenue Adolphe Chauvin, Cergy-Pontoise Cedex, 95302, France.
Gallet
O
O
Rouahi
M
M
Imhoff
J M
JM
D011232:Chemical Precipitation; D002846:Chromatography, Affinity; D004355:Drug Stability; D005353:Fibronectins / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification*; D005780:Gelatin; D006493:Heparin; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques
D005353:Fibronectins; D005780:Gelatin; D006493:Heparin