Performing a Separation of Fibronectin with Gelatin Sepharose 4B

Extracted from Affinity Chromatography Principles and Methods, GE Healthcare, 2007

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.

Purification Option

  Binding capacity/ml medium Maximum operating flow Comments
Gelatin Sepharose 4B 1 mg human plasma fibronectin 75 cm/h* Supplied as a suspension ready for column packing.

* See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.

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.

Media Characteristics

  Ligand density Composition pH stability* Mean particle size
Gelatin Sepharose 4B 4.5–8 mg gelatin/ml Gelatin linked to Sepharose  using the CNBr method Short term 3–10 Long term 3–10 90 µm

* Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures.

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.

Materials

     

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