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Fragmentation of IgG using pepsin
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Materials required

 

  • Chromatographic column
  • pH meter
  • Magnetic stirrer
  • Visi cooler
  • Pasteur pipette
  • Centrifuge
  • Gel filtration column
  • Spectrophotometer
  • Pepsin
  • IgG antibody
  • PBS buffer
  • 0.1M sodium acetate buffer
  • Sephacryl S-200
  • Glycerol

 

Procedure

 

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

 

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

 

  • Mix the contents in the tube by proper shaking.

 

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

 

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

 

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

 

  • Incubate the antibody at 37oC for overnight.

 

  • Again adjust the pH to 7.4 after incubation.

 

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

 

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

 

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

 

  • Determine A280 by Spectrophotometric analysis.

 

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

 

 

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