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Fragmentation of IgG Using Papain
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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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