Purification of IgG Antibodies using Affinity Chromatography
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.