Affinity Purificatoin of Antibodies (Coupling to Activated CH Sepharose 4B)
From Bridges Lab Protocols
- 0.1M NaBicarbonate (0.8401g in 100mL ddH2O) and 0.5MNaCl (2.922g in 100mL ddH2O)at pH 8.0.
- 50mM Sodium Acetate (0.41g in 100mL ddH2O)at pH 4.0
- 50mM Tris (0.61g in 100mL ddH2O) and .5M NaCl (2.922g in 100mL ddH2O) at pH 8.0
- 0.4M glycine (3.004g in 100mL ddH2O)at pH 1.8.
Protein must be extensively dialysed in PBS to remove any amine group from the sample.
- Swell 0.5g of CH Sepharose 4B in 200 mL of ice cold 1 mM HCl for 15 minutes. Gives ~1.5 mL of Sepharose.
- Wash separose with coupling buffer (1.0M NaHCO3), 0.5 M NaCl, pH 8.0)
- Dissolve protein ligand (~1mg) in 6 mL of coupling buffer.
- Mix sepharose and ligand end over end at room temperature for 2-4 hours.
- Retain flow through for analysis and wash sepharose with coupling buffer.
- Block Sepharose with 1 M Tris/HCl, pH 8.0 for 1 hour end over end at room temperature. Alternatively block overnight at 4oC.
- Wash sepharose with 3 cycles of 50mM sodium acetate, 0.5 M NaCl, pH 4.0 the 50 mM Tris/HCl, 0.5 M NaCl, pH 8.0.
- Store in coupling buffer with 0.02% sodium azide.
- Add 10 uL of flow through to 1 mL of Bradford's Reagent.
- Coupling % should be between 70 and 80%.
- For Peptides use 5 mg in 1 ml of coupling buffer. Check pH has not changed. Read OD241 before and after coupling for coupling efficiency.
- Dialysis of 10 mL sera against PBS for 1-2 hours at 4oC.
- Wash Sepharose with 2 column volumes of 50 mM Tris/HCl, pH 7.5 with 0.5 M nACl then 50 mM Tris/HCl, pH7.5.
- Dilute sera with equal volume of 50 mM Tris/HCl, pH 7.5.
- Load onto column end over end for 2-4 hours at 4oC.
- If column flow through needs to be kept store at -80oC.
- Wash sepharose with 50 mM Tris/HCl, pH 7.5 with 0.5 M NBaCl.
- Pack into Bio-rad plastic column. Wash until eluent is clean.
- Elute antibodies with 100 mM glycine pH 1.8. Collect 10 x 1 mL fractoins. Bring pH of antibodies back to neutral by adding ~250 uL of 1 M Tris/HCl, pH 8.0.
- Assay for protein and pool the protein peak.
- Dialysis against 1 L of PBS for 2-4 hours and 2 L of PBS overnight at 4oC.
- Concentrate sample, assay for protein and aliquot antibodies. Store at -80oC.
- Store column in 50 mM Tris/HCl, pH 7.5 with sodium azide at 4oC.