Materials required

1. Chromatographic column

2. pH meter

3. Magnetic stirrer

4. Visi cooler

5. Pasteur pipette

6. Centrifuge

7. Gel filtration column

8. Spectrophotometer

9. Pepsin

10. IgG antibody

11. PBS buffer

12. 0.1M sodium acetate buffer

13. Sephacryl S-200

14. Glycerol

     

Procedure

1. Take 10ml of PBS buffer in a 15ml screw capped tube.

2. Weigh out 0.2g of antibody and added to the tube.

3. Mix the contents in the tube by proper shaking.

4. Dialyzed the IgG antibody against 0.1M sodium acetate buffer for 3 hours.

5. After dialysis, adjust the pH of the dialyzed antibody to 4.5 from 8.00 by adding acetic acid.

6. Add 4mg of pepsin to the dialyzed antibody. And mix well.

7. Incubate the antibody at 37oC for overnight.

8. Again adjust the pH to 7.4 after incubation.

9. Centrifuge the antibody-pepsin solution at 1000g for 15 min.

10. After centrifugation, the precipitate formed in the solution is discarded using a pipette.

11. Fractionate the mixture by gel filtration on a column of Sephacryl S-200 in PBS buffer.

12. Determine A280 by Spectrophotometric analysis.

13. Store the purified antibody fragments at -20 oC with Glycerol added at a final concentration of 50%.