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