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Affinity Purification

Affinity Purification

Serum Purification:

  1. Wash column with 10-15 ml of 0.01M PBS (pH = 7.2)
  2. Mix 1 ml of serum with 1 ml of 0.01M PBS.
  3. Add the diluted serum to the gel column and let this drain into the gel.
  4. Cap the bottom of the column and let incubate for 1 hour at room temperature.
  5. Wash the column with 10-15 ml of 0.01M PBS.
  6. Add 1 ml of Elution Buffer (100 mM Glycine Buffer, pH=2.8) and collect in an eppendorf container.  Do this about 6-8 times collecting (6) 1 ml samples of the eluted buffer.  In each eppendorf container add 50µl of 1M Tris.
  7. Test each ml of elution buffer collected by coating microwell with 10µl of Bradford Assay and adding 50µl of eluted buffer.  This will help you determine which 1 ml samples have the desired antibody.
  8. Dialyze what you have collected and kept overnight in 4 liters of 1 X PBS.
  9. Take OD reading off spectrometer at 280 nm.  The extinction coefficient is 1.4.
  10. When finished with column, wash with 10-15ml of Sodium Azide and store at 4°C with ~4 ml of Sodium Azide in gel column.