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'''Procedure'''
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.
