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.

Chromatography medium characteristics

The characteristics of Gelatin Sepharose 4B chromatography medium are shown in Table 3.13.

Table 3.13. Characteristics of Gelatin Sepharose 4B chromatography medium

  Ligand density (mg/ml) Composition pH stability1 Average
Gelatin Sepharose 4B 4.5 to 8.0 Gelatin coupled to Sepharose 4B using the CNBr method. Short term: 3 to 10
Long term: 3 to 10
90

1 Short term refers to the pH interval for regeneration, cleaning-in-place, and sanitization procedures. 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.

Purification option

Gelatin Sepharose 4B is available in chromatography media packs for packing into columns of your choice (Table 3.14).

Table 3.14. Purification option for Gelatin Sepharose 4B

  Binding capacity/ml medium Maximum operating flow (cm/h)1 Comments
Gelatin Sepharose 4B Human plasmafi bronectin, 1 mg 75 Supplied as a suspension ready for column packing.

1 See Appendix 4 to convert flow velocity (cm/h) to volumetric flow rate (ml/min). 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: 50 mM sodium acetate, 1 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 three times with 2 to 3 CV of buffer, alternating between high pH (100 mM Tris-HCl, 500 mM NaCl,pH 8.5) and low pH (100 mM sodium acetate, 500 mM NaCl, pH 4.5). Re-equilbrate immediately with 3 to 5 CV of binding buffer. Remove denatured proteins or lipids by washing the column with 0.1% Tween 20 at 37°C for 1 min. Re-equilibrate immediately with 5 CV of binding buffer.

Chemical stability

Stable in all commonly used aqueous buffers.

Storage

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

Materials