Difference between revisions of "Affinity Purificatoin of Antibodies (Coupling to Activated CH Sepharose 4B)"
From Bridges Lab Protocols
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| − | + | ==Materials== | |
#0.1M NaBicarbonate (0.8401g in 100mL ddH<sub>2</sub>O) and 0.5MNaCl (2.922g in 100mL ddH<sub>2</sub>O)at pH 8.0. | #0.1M NaBicarbonate (0.8401g in 100mL ddH<sub>2</sub>O) and 0.5MNaCl (2.922g in 100mL ddH<sub>2</sub>O)at pH 8.0. | ||
#50mM Sodium Acetate (0.41g in 100mL ddH<sub>2</sub>O)at pH 4.0 | #50mM Sodium Acetate (0.41g in 100mL ddH<sub>2</sub>O)at pH 4.0 | ||
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#0.4M glycine (3.004g in 100mL ddH<sub>2</sub>O)at pH 1.8. | #0.4M glycine (3.004g in 100mL ddH<sub>2</sub>O)at pH 1.8. | ||
| − | + | ==Procedure== | |
| − | Protein must be extensively dialysed in | + | 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. | #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) | #Wash separose with coupling buffer (1.0M NaHCO<sub>3</sub>), 0.5 M NaCl, pH 8.0) | ||
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#Store in coupling buffer with 0.02% sodium azide. | #Store in coupling buffer with 0.02% sodium azide. | ||
| − | + | ==Coupling Efficiency== | |
| − | Add 10 uL of flow through to 1 mL of Bradford's Reagent. | + | *Add 10 uL of flow through to 1 mL of Bradford's Reagent. |
| − | Coupling % should be between 70 and 80%. | + | *Coupling % should be between 70 and 80%. |
| − | + | *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. | |
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| − | + | ==Affinity Purification== | |
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#Dialysis of 10 mL sera against PBS for 1-2 hours at 4<sup>o</sup>C. | #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. | #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. | ||
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.
- 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.
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
- 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.
