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 37^oC 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%.

 

 

Help:

 

                    Unfortunately, this Virtual Lab requires Adobe Flash player.  Please see additional information if this does not work on your computer

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• For Microsoft Edge - https://support.microsoft.com/en-in/help/4532571/microsoft-edge-turn-on-flash 

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On GNU/Linux machines, please see appropriate online help. For example, 

 

• on Ubuntu - https://help.ubuntu.com/stable/ubuntu-help/net-install-flash.html.en 

• on Fedora - https://docs.fedoraproject.org/en-US/quick-docs/using-adobe-flash/