Rapid, high-yield method for the bulk purification of fibronectin from human plasma.

Fibronectin is an abundant, high molecular-weight, extracellular glycoprotein that exists in a soluble form in body fluids and in an insoluble form in the extracellular matrix (3,4). Fibronectin plays a major role in many important physiological processes, such as embryogenesis, wound healing, hemostasis and thrombosis. It is a highly affinity-adhesive protein that binds to numerous matrix macromolecules, including collagen or gelatin, fibrin (ogen), heparin and other glycosaminoglycans, as well as cell surface receptors. Fibronectin has been used extensively in tissue culture as a reagent that effectively adheres cells to various substrata. However, fibronectin is a relatively costly reagent, and it is difficult for many laboratories to prepare it themselves. A low-cost and high-yield method for the isolation of pure fibronectin would significantly reduce the cost of performing many types of tissue culture. Here, we describe a procedure for producing chromatographically pure fibronectin using an inexpensive and simple process based on gelatin bead affinity chromatography. Gelatin beads were prepared using a modification of the procedure described by Atkanov et al. (1). Sunflower oil (300 mL) was preheated to 50°C in a pre-siliconized, flat-bottomed glass flask with a 7 cm stir bar. This mixture was spun at 200 rpm. Then, 70 μL of Triton X-114 was added to the oil, and the sample was stirred for 5 min. An aqueous phase containing 12 g of porcine skin gelatin (Sigma, St. Louis, MO, USA) dissolved in 60 mL of deionized water (also preheated to 50°C) was added to the oil. The rotation speed was increased to approximately 500 rpm, and the mixture was stirred for 30 min. Alternatively, the oil and gelatin mixture was placed in a tabletop blender and the mixture blended on the lowest setting for 5 min. This oil and gelatin mixture was then cooled immediately to 18°C by immersion in an ice/water bath. The beads were separated from the oil emulsion by sedimentation as follows: 500 mL PBS (pH 7.3), containing 0.2% Triton X-100 was added slowly to the cooled emulsion and stirred for another 10 min. The gelatin beads were allowed to settle out of the emulsified oil phase for at least 1 h. Often, this is a convenient overnight stopping point for the procedure. The remaining supernatant oil was then removed, and the beads were washed 3× with cold (4°C) deionized water. The sedimentation time of the beads was 10 min between washes. The beads were then stabilized by glutaraldehyde fixation. The beads were suspended in 500 mL of PBS containing 2 mL electron microscopy-grade glutaraldehyde (25%, Sigma) and the solution stirred for an additional hour at room temperature. The beads were then washed 3× with deionized water. The beads prepared in this manner were used for conventional fibronectingelatin affinity chromatography as previously described (1). The following procedures were performed at room temperature. Blood from normal nonmedicated adults (50 mL) was collected by venipuncture using heparin as an anticoagulant. The blood was centrifuged at 1000× g in a Sorvall-2B centrifuge (Dupont, Wilmington, DE, USA) for 30 min to separate plasma from packed red cells. The plasma was supplemented with PMSF in ethanol to a final concentration of 44 μg per every 25 mL of plasma (44 μL of a 1 mg/mL solution in absolute ethanol). Plasma plus PMSF (25 mL) was added to 25 mL of gelatin beads and rocked overnight (12 h) at room temperature. Twenty-five milliliters of beads mixed with 25 mL of treated plasma were applied to a column made of a 50 mL pipet plugged with 1 cm of glass wool and 1 cm of washed sand. The column was perfused with approximately 10 column volumes of PBS (250–300 mL) to wash off contaminating plasma proteins, mainly albumin. After the column had been washed thoroughly, the fibronectin bound to the beads was eluted with 4 M urea, 50 mM Tris (pH 7.5). We collected fifty 5 mL fractions on a fraction collector. The protein content in each sample was determined by sampling 10 μL from each tube to determine the peak protein fractions from the column (Figure 1). The protein-rich fractions were pooled (approximately 50 mL) and immediately dialyzed against 2 L of PBS to remove the urea. Dialysis was carried out twice to reduce urea to negligible levels. Spectrapor cellulose dialysis memBenchmarks