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

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'''Procedure'''
 
'''Procedure'''
 
Protein must be extensively dialysed in PPBS to remove any amine group from the sample.  
 
Protein must be extensively dialysed in PPBS 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 NaHCO<sub>3</sub>), 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 4<sup>o</sup>C.
 +
#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.
 +
 
 +
'''Coupling Efficiency'''
 +
Add 10 uL of flow through to 1 mL of Bradford's Reagent.
 +
Coupling % should be between 70 and 80%.
 +
{| border="1" cellspacing="0" cellpadding="5" align="center"
 +
| Average OD<sub>595</sub>
 +
|           
 +
|-
 +
| mg/mL
 +
|         
 +
|-
 +
|Total Volume
 +
|           
 +
|-
 +
|Total mg
 +
|         
 +
|-
 +
|% Coupling
 +
|       
 +
|-
 +
|}
 +
 
 +
For Peptides use 5 mg in 1 ml of coupling buffer. Check pH has not changed. Read OD<sub>241</sub> before and after  coupling for coupling efficiency.
 +
 
 +
'''Affinity Purification'''
 +
#Dialysis of 10 mL sera against PBS for 1-2 hours at 4<sup>o</sup>C.
 +
#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 4<sup>o</sup>C.
 +
#If column flow through needs to be kept store at -80<sup>o</sup>C.
 +
#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 4<sup>o</sup>C.
 +
#Concentrate sample, assay for protein and aliquot antibodies. Store at -80<sup>o</sup>C.
 +
#Store column in 50 mM Tris/HCl, pH 7.5 with sodium azide at 4<sup>o</sup>C.

Revision as of 13:52, 20 May 2009

Materials

  1. 0.1M NaBicarbonate (0.8401g in 100mL ddH2O) and 0.5MNaCl (2.922g in 100mL ddH2O)at pH 8.0.
  2. 50mM Sodium Acetate (0.41g in 100mL ddH2O)at pH 4.0
  3. 50mM Tris (0.61g in 100mL ddH2O) and .5M NaCl (2.922g in 100mL ddH2O) at pH 8.0
  4. 0.4M glycine (3.004g in 100mL ddH2O)at pH 1.8.

Procedure Protein must be extensively dialysed in PPBS 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 4oC.
  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%.

Average OD595
mg/mL
Total Volume
Total mg
% Coupling

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 4oC.
  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 4oC.
  5. If column flow through needs to be kept store at -80oC.
  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 4oC.
  11. Concentrate sample, assay for protein and aliquot antibodies. Store at -80oC.
  12. Store column in 50 mM Tris/HCl, pH 7.5 with sodium azide at 4oC.
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