Affinity Purification Protocol
Affinity Purification
Serum Purification:
- Wash column with 10-15 ml of 0.01M PBS (pH = 7.2)
- Mix 1 ml of serum with 1 ml of 0.01M PBS.
- Add the diluted serum to the gel column and let this drain into the gel.
- Cap the bottom of the column and let incubate for 1 hour at room temperature.
- Wash the column with 10-15 ml of 0.01M PBS.
- 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.
- 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.
- Dialyze what you have collected and kept overnight in 4 liters of 1 X PBS.
- Take OD reading off spectrometer at 280 nm. The extinction coefficient is 1.4.
- 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.
Print
10/12/2007