Biotechnology and Biomedical Engineering Immunology Virtual Lab II Experiments

Purification of IgG Antibodies with Ammonium Sulphate

Materials Required

  1. PBS
  2. Saturated ammonium sulfate
  3. Ascites fluid or MAb supernatant
  4. Refrigerated centrifuge
  5. Dialysis tubing and clamp
  6. Magnetic stirrer and Magnetic stir bar
  7. Absorbent paper
  8. Forceps
  9. Glass wool and glass rod
  10. Centrifuge tube
  11. Pasteur pipette
  12. Pipette pump
  13. Glass pipette
  14. Funnel
  15. Visi cooler

 

Reagent Preparation

 

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.

 

Procedure

  1. 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.

  2. 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.

  3. Decant the supernatant by pipetting out with a Pasteur pipette into a 25 ml beaker.

  4. Place the beaker ( 25 ml) containing the ascites on ice tray and place that over a magnetic stirrer.

  5. 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.

  6. 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.

  7. Dissolve precipitate in a 10 ml volume of PBS and resuspend the pellet using a glass rod.

  8. Using forceps takeout dialysis tubing from ethanol storage solution and rinse dialysis tubing with distilled water into wash beaker.

  9. 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.

  10. Place dialysis tubing in a beaker containing PBS buffer.

  11. 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.

  12. Change the dialysis buffer four times during dialysis.

 

Result

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.