- Saturated ammonium sulfate
- Ascites fluid or MAb supernatant
- Refrigerated centrifuge
- Dialysis tubing and clamp
- Magnetic stirrer and Magnetic stir bar
- Absorbent paper
- Glass wool and glass rod
- Centrifuge tube
- Pasteur pipette
- Pipette pump
- Glass pipette
- Visi cooler
- Saturated Ammonium Sulfate (SAS): Weigh 76 g ammonium sulfate and dissolve in 100 ml water .Heat with stirring to just below boiling point. Leave overnight at room temperature (The solubility of ammonium sulfate at 1000C is 76 g per 100 ml).
- 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 (7.2 to 7.4) using 1 M NaOH or 1 M HCl. Add water to get a final volume of 1 litre.
- Remove the lipid content of ascites fluid by placing enough glass wool into a funnel to cover the opening. Pour ascites through the funnel and the filtered sample is collected in a beaker. Rinse the glass wool with PBS (phosphate buffered saline) and squeeze glass wool gently with glass rod to obtain the entire sample.
- Transfer the sample to a centrifuge tube and centrifuge filtered ascites 30 min at 20,000 × g (13,000 rpm in SS-34 rotor) either 4°C or room temperature.
- Decant the supernatant by pipetting out with a Pasteur pipette into a 25 ml beaker.
- Place the beaker ( 25 ml) containing the ascites on ice tray and place that over a magnetic stirrer.
- Add 4 ml saturated ammonium sulfate slowly by means of a pipette. Leave 1 to 2 hr at 4°C to ensure precipitation of all the protein.
- Transfer the solution to centrifuge tube and Centrifuge 1 hr at 20,000 × g, either 4°C Decant off the supernatant solution to beaker, while pellet retains in the centrifuge tube.
- Dissolve precipitate in a 10 ml volume of PBS and resuspend the pellet using a glass rod.
- Using forceps takeout dialysis tubing from ethanol storage solution and rinse dialysis tubing with distilled water into wash beaker.
- Clamp one end of the tubing using a dialysis clamp. Fill the dissolved precipitate to approximately one half of the capacity and close the tubing with a clamp.
- Place dialysis tubing in a beaker containing PBS buffer.
- Dialyze at least 3 hour at the desired temperature ( 4 degree Celsius ) with gentle stirring of the buffer – Place the beaker on magnetic stirrer and place the stir bar in the beaker.
- Change the dialysis buffer four times during dialysis.
The IgG antibodies percipitated with ammonium sulphate is purified by Dialysis using PBS buffer and the purified IgG antibodies is stored at 4 degree celsius.
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