Performing a Separation of Fibronectin with Gelatin Sepharose^® 4B

Fibronectin is a high molecular weight glycoprotein found on the surfaces of many cell types and present in many extracellular fluids including plasma. Fibronectin binds specifically to gelatin at or around physiological pH and ionic strength.

Performing a Separation

Binding buffer: PBS: 140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4

Elution buffer alternatives:

• 0.05 M sodium acetate, 1.0 M sodium bromide (or potassium bromide), pH 5.0
• Binding buffer + 8 M urea
• Binding buffer + arginine

Fibronectin has a tendency to bind to glass. Use siliconized glass to prevent adsorption.

Cleaning

Wash 3 times with 2–3 column volumes of buffer, alternating between high pH (0.1 M Tris-HCl,  0.5 M NaCl, pH 8.5) and low pH (0.1 M sodium acetate, 0.5 M NaCl, pH 4.5). Re-equilbrate immediately with 3–5 column volumes of binding buffer. Remove denatured proteins or lipids by washing the column with 0.1% Triton X-100 at +37 °C for one minute. Re-equilibrate immediately with 5 column volumes of binding buffer.

Chemical Stability

Stable in all commonly used aqueous buffers.

Storage

Wash media and columns with 20% ethanol at neutral pH (use approximately 5 column volumes for packed media) and store at +4 to +8 °C.