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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