||3 ml prepacked column, or 5 ml prepacked column|
|6% highly cross-linked spherical agarose|
||Recombinant Vascular Endothelial Growth Factor|
| 300 cm/h|
Recommended Linear Flow Rate:
|50~150 cm/h |
| pH 2.5~9|
| 4 °C to 8 °C in 20% ethanol|
VEGF was immobilized onto solid supports of sepharose beads for screening compounds interacting with VEGF.
Protocol of Capturing Molecules Interacting with VEGF165
- Wash the prepacked 5 ml column with 5~10 column volumes of distilled water to remove 20% ethanol.
- Equilibrate the prepacked 1 ml column with 5~10 column volumes of Binding Buffer: 20 mM sodium phosphate, 150 mM NaCl, pH 7.4.
- Filtrate 3-5 ml target samples through a 0.45 μm filter and load the sample.
- Wash with 5-10 column volumes of Binding Buffer: 20 mM sodium phosphate, 150 mM NaCl, pH 7.4.
- Wash with 10 column volumes of elution buffer I (100 mM sodium citrate, pH 6.0) and followed by 10 column volumes of elution buffer II (100 mM sodium glycine, pH 2.7).
- Discard first 1/3 column volume of elution buffer II. Immediately collect 1 column volume of eluant by elution buffer II. Neutralize collected fractions with neutralization buffer (1M Tris-HCl, pH 9.0).
- HSA affinity column was used as a control.
- Analyze the elute fraction by HPLC or other chromatography techniques to ensure that target molecule is captured.
- Use the prepacked column for easier process.
- The target molecule should be stable under the selected elution conditions.
- Avoid air bubbles in the column.
- It is recommended to regenerate the resin by 4 column volumes of 0.1 M glycine-HCl (pH 2.7) after 5 separation cycles.
- After 10 separation cycles, wash the resin with 5 column volumes of 20% ethanol and 5~10 column volumes of 70% ethanol sequentially to remove hydrophobic substances.
- For longer periods of storage, keep at 4~8ºC in 20% ethanol.
|For research purposes only. Not for human or animal therapeutic or diagnostic use.|