Affinity Chromatography - Department of Molecular and Cellular ...

Purification or removal of fibronectin
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.
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 Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , 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 4.5–8 mg gelatin/ml Gelatin linked to Sepharose Short term 3–10 90 µm
Sepharose 4B using the CNBr method Long term 3–10
*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.
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