Difference between revisions of "Affinity Purificatoin of Antibodies (Coupling to Activated CH Sepharose 4B)"

Latest revision as of 00:15, 27 May 2009

Materials

• 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.

Procedure

Protein must be extensively dialysed in PBS to remove any amine group from the sample.

1. 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.
2. Wash separose with coupling buffer (1.0M NaHCO₃), 0.5 M NaCl, pH 8.0)
3. Dissolve protein ligand (~1mg) in 6 mL of coupling buffer.
4. Mix sepharose and ligand end over end at room temperature for 2-4 hours.
5. Retain flow through for analysis and wash sepharose with coupling buffer.
6. Block Sepharose with 1 M Tris/HCl, pH 8.0 for 1 hour end over end at room temperature. Alternatively block overnight at 4^oC.
7. 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.
8. Store in coupling buffer with 0.02% sodium azide.

Coupling Efficiency

• 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.

Affinity Purification

1. Dialysis of 10 mL sera against PBS for 1-2 hours at 4^oC.
2. 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.
3. Dilute sera with equal volume of 50 mM Tris/HCl, pH 7.5.
4. Load onto column end over end for 2-4 hours at 4^oC.
5. If column flow through needs to be kept store at -80^oC.
6. Wash sepharose with 50 mM Tris/HCl, pH 7.5 with 0.5 M NBaCl.
7. Pack into Bio-rad plastic column. Wash until eluent is clean.
8. 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.
9. Assay for protein and pool the protein peak.
10. Dialysis against 1 L of PBS for 2-4 hours and 2 L of PBS overnight at 4^oC.
11. Concentrate sample, assay for protein and aliquot antibodies. Store at -80^oC.
12. Store column in 50 mM Tris/HCl, pH 7.5 with sodium azide at 4^oC.