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'''==Materials'''==
'''==Procedure'''==Protein must be extensively dialysed in PPBS PBS to remove any amine group from the sample.
'''==Coupling Efficiency'''==*Add 10 uL of flow through to 1 mL of Bradford's Reagent.*Coupling % should be between 70 and 80%.{| border="*For Peptides use 5 mg in 1" cellspacing="0" cellpadding="5" align="center"| Average ml of coupling buffer. Check pH has not changed. Read OD<sub>595241</sub>| |-| mg/mL| |-|Total Volume| |-|Total mg| |-|% Coupling| |-|}before and after coupling for coupling efficiency.
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'''==
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#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
#0.4M glycine (3.004g in 100mL ddH<sub>2</sub>O)at pH 1.8.
#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)
#Store in coupling buffer with 0.02% sodium azide.
#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.