Materials Required:

 

• IgG Antibody                                                      
• Papain
• 0.1M cysteine solution
• 0.5M EDTA solution
• 0.01M sodium acetate buffer 
• PBS Buffer
• Ethanol storage solution
• 50% Glycerol solution
• CM-Cellulose slurry
• DEAE-Cellulose slurry
   

 

Procedure:
 

Preparation of antibody-papain solution

 

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

 

Dialysis of  antibody –papain solution

 

• 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.
• Pipette out the entire antibody -Papain solution from the vial and added to the dialysis tubing.
• 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.
• Immerse this dialysis tubing in a beaker or flask containing 0.01M Sodium acetate Buffer.
• 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.
• Next day, take out the beaker  from the Visicooler.
• 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
 

•  Wash the chromatographic column for 2 times using distilled water.
• Then the chromatographic column is prepared  using CM-Cellulose Slurry.
• Saturate the CM-Cellulose slurry by 0.01M sodium acetate buffer.
• After saturation, the sample solution is added to the column.
• 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 
 

 

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

Dialysis of purified sample 2 in PBS buffer

 

• 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.
• Pipette out the entire Purified sample 2 from the tube and added to the dialysis tubing.
• 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.
• Immerse this dialysis tubing in a beaker or flask containing PBS  Buffer.
• 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.
• Next day, take out the beaker  from the Visicooler.
• 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.
• Label the tube as Purified Fab Fragments.
  
   

 Glycerol Stock Preparation

 

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

 

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