Materials Required:

1. IgG Antibody
2. Papain
3. 0.1M cysteine solution
4. 0.5M EDTA solution
5. 0.01M sodium acetate buffer
6. PBS Buffer
7. Ethanol storage solution
8. 50% Glycerol solution
9. CM-Cellulose slurry
10. DEAE-Cellulose slurry

Procedure:

Preparation of antibody-papain solution

1. Take 5 ml of PBS buffer in a 15ml screw capped tube.
2. Weigh 0.1g of antibody and added to the tube and mix it well.
3. Pipette out 1ml of 0.1M cysteine solution to the tube.
4. Pipette out 20 microlitre of 0.5 M EDTA solution and added to the tube.
5. Weigh 1mg of Papain and added to the tube.
6. Mix the contents in the tube by proper shaking.
7. Incubate the antibody-Papain solution at 370c for 4 hours.

Dialysis of antibody –papain solution

1. Take a piece of dialysis tubing from ethanol storage solution with forceps and washed it with distilled water and put one clamp on one side of the tubing.

2. Pipette out the entire antibody -Papain solution from the vial and added to the dialysis tubing.

3. The air is removed by gently squeezing the tube by sliding fingers all the way up the tube, and put second clamp on the other end of the tubing.

4. Immerse this dialysis tubing in a beaker or flask containing 0.01M Sodium acetate Buffer.

5. Put the stir bar in the beaker and the beaker was placed on a magnetic stirrer and keep inside of Visicooler at 4°C for overnight incubation.

6. Next day, take out the beaker from the Visicooler.

7. The dialysis tubing has to be taken from the beaker and content in the tube was transferred to a centrifuge tube using a Pasteur pipette.

    

Fractionation of dialysed antibody-papain solution on CM-Cellulose slurry

1. Wash the chromatographic column for 2 times using distilled water.
2. Then the chromatographic column is prepared using CM-Cellulose Slurry.
3. Saturate the CM-Cellulose slurry by 0.01M sodium acetate buffer.
4. After saturation, the sample solution is added to the column.
5. Collect the fractionated sample to a new sterile tube and label the tube as Purified sample 1.

Refractionation of purified sample1 on DEAE –Cellulose slurry

1. Wash the chromatographic column for 2 times using distilled water.
2. Then the chromatographic column is prepared using DEAE -Cellulose Slurry.
3. Saturate the DEAE-Cellulose slurry by 0.01M sodium acetate buffer.
4. After saturation, the sample solution is added to the column.
5. Collect the refractionated sample to a new sterile tube and label the tube as Purified sample 2.
6. The purified sample is subjected to UV-Spectrophotometric Analysis.

Dialysis of purified sample 2 in PBS buffer

1. Take a piece of dialysis tubing from ethanol storage solution with forceps and washed it with distilled water and put one clamp on one side of the tubing.
2. Pipette out the entire Purified sample 2 from the tube and added to the dialysis tubing.
3. The air is removed by gently squeezing the tube by sliding fingers all the way up the tube, and put second clamp on the other end of the tubing.
4. Immerse this dialysis tubing in a beaker or flask containing PBS Buffer.
5. Put the stir bar in the beaker and the beaker was placed on a magnetic stirrer and keep inside of Visicooler at 4°C for overnight incubation.
6. Next day, take out the beaker from the Visicooler.
7. The dialysis tubing has to be taken from the beaker and content in the tube was transferred to a centrifuge tube using a Pasteur pipette.
8. Label the tube as Purified Fab Fragments.

Glycerol Stock Preparation

1. Pipette out 5ml of 50% glycerol and added to a cryovial.
2. Pipette out 5ml of purified Fab fragment solution from the tube and added to the tube.
3. The cryovial is wrapped at the upper portion using Para film.
4. The wrapped cryovial is then placed in the cryobox.
5. The cryobox containing the vial is placed on the -200 freezer.