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Purification of IgG Antibodies using Affinity Chromatography
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MATERIALS REQUIRED:

 

  • Ascites fluid or MAb supernatant
  • PBS Buffer
  • Tris base buffer
  • Citric acid
  • Column
  • Dialysis tubing and clamps
  • Magnetic stirrer and Magnetic stir bar
  • Absorbent paper
  • Forceps
  • Glass pipette
  • Pipette pump
  • Refrigerated centrifuge
  • Centrifuge tube
  • 0.45µm filter
  • Syringe
  • Spectrophotometer
  • Pasteur pipette
  • Test tubes

 

REAGENT PREPARATION:

 

  • PBS (phosphate-buffered saline): Weigh  0.23 g NaH2PO4 (anhydrous; 1.9 mM),1.15 g NaH2PO4 (anhydrous; 8.1 mM), 9.00 g NaCl (154 mM).Add water to a volume  of  900 ml. Adjust to desired pH  using 1 M NaOH or 1 M HCl. Add water to get a final volume of 1 liter.

 

  • HCl ( 1 M ) : Mix in the following order:  913.8 ml H2O  and 86.2 ml concentrated HCl.

 

  • NaOH (10 M) : Dissolve 400 g NaOH in 450 ml H2O. Add H2O to 1 liter.

 

PROCEDURE:

 

  • Centrifuge monoclonal antibody supernatant at 20,000 × g (13,000 rpm in SS-34 rotor)  at 4°C.

 

  • Filter the supernatant by filtering  through a 0.45-μm filter.

 

  • Adjust MAb supernatant to pH 8.0 by dialysis against PBS, pH 8.0 by using dialysis tubing.

 

  •  Prepare protein A–Sepharose column and attach to fraction collector.

 

  • Equilibrate column with PBS, pH 8.0, at either 4°C or room temperature.

 

  • Layer antibody solution onto resin bed.

 

  • Wash column with several volumes PBS, pH 8.0.

 

  • Elute with 0.1 M citric acid at suitable pH 3 (bring to appropriate pH with 1 M NaOH): for mouse IgG1 use pH 6.5, for IgG2a use pH 4.5, and for IgG2b and IgG3 use pH 3.0.

 

  • Collect the eluent in vials and dialyze elutes  against PBS, pH 7.3.

 

  • Change the dialysis buffer twice. Store samples in PBS or borate-buffered saline at 4°C.

 

 

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