Flash content link: https://vlab.amrita.edu/repo/BIOTECH/IMM/Extraction_of_IgG_Antibodies_From_Immunized_Hen_Egg/index.swf
Materials Required:
- Immunized hen egg
- TBS
- Dextran sulphate solution
- Calcium chloride solution
- Saturated sodium sulphate solution
- 2% Sodium bicarbonate solution
- 1mM EDTA
- 005 M Tris Hcl Buffer,pH = 8
- PBS Buffer
- Ethanol storage solution
- DEAE cellulose slurry
- Glycerol
- Distilled water
- Magnetic Stirrir
- Stir Bar
- Visicooler
- Dialysis tubing
- Chromatographic column
- UV spectrophotometer
- Cryovial
- Cryobox
- -200 CFreezer
Procedure:
- Sterilize the egg shell with 70% ethanol.
- The yolk is separated from the white and the yolk membrane is cut open.
- Pour the yolk into a sterile beaker and dilute it with 1000 µl of Tris-buffered saline (TBS).
- Transfer the diluted egg yolk into a sterile centrifuge tube. Centrifuge at 5000 rpm for 10 minutes.
- Pipette the precipitate carefully from the centrifuge tube.
- Add 1000 µl of Dextran Sulphate solution into diluted egg yolk suspension.
- Then add 1000 µl of Calcium chloride solution into the diluted egg yolk and mix it well.
- Pipette the precipitate carefully from the centrifuge tube.
- Again add 4000 µl of Tris-buffered saline into the diluted egg yolk suspension.
- Centrifuge the suspension at 5000 rpm for 10 minutes.
- After centrifugation, carefully collect the supernatant into a new beaker. Put a magnetic stirrer (stir bar) in the beaker.
- Add 4000 µl of Sodium sulphate into the supernatant solution.
- Keep the supernatant in the visicooler for 2 hours at 4oC. Mix the supernatant well with the magnetic stir bar.
- Carefully remove the stir bar from the beaker.
- Transfer the supernatant to a centrifuge tube labeled concentrated IgG.
- Centrifuge the suspension at 13000 rpm for 1 hour at 4oC.
- After centrifugation carefully discard the supernatant and the pellet is collected at the bottom of the centrifuge tube.
- Add 10 ml of TBS buffer to the pellet and mix it properly. Keep the centrifuge in the ice box.
Purification of IgG Fraction by Dialysis Activation of Dialysis Tubing:
- Place a small piece of dialysis tubing in a beaker containing 2% sodium bicarbonate solution. Keep it in the water bath for 20 minutes at 100oC.
- After that, take the dialyzing tube from the sodium bicarbonate solution and wash it with water.
- Keep the dialyzing tube in 1Mm EDTA. Keep it in the water bath for 20 minutes at 100oC.
- Then take the dialyzing tube from 1Mm EDTA and wash it with water.
- Rinse the dialyzing tube with 005 M Tris Hcl buffer (pH =8).
- Place the dialyzing tube on a tissue paper.
Purification of IgG Antibodies:
- Pipette 10 ml of the IgG pellets suspended in TBS buffer.
- Add the resuspended solution into an activated dialyzing tube and clamp it properly.
- Wipe the dialyzing tube with tissue paper.
- Place the activated dialyzing tube in a beaker containing PBS buffer (pH =7.2). Keep a magnetic stirrer in the beaker.
- Place the beaker in a Visicooler for 3 hours at 4oC. Mix the supernatant well with the magnetic stir bar.
- Then, pipette the IgG suspension in the dialyzing tubing using a sterile pasteur pipette. Transfer the IgG suspension into a new centrifuge tube labeled dialyzed IgG.
Purification of IgG Fraction by Ion-Exchange Chromatography:
- Carefully wash the chromatographic column with distilled water.
- Transfer DEAE cellulose slurry into the chromatographic column. Then transfer PBS buffer into the column. Later collect PBS buffer into a beaker and then tap the column properly.
- Transfer the dialysate IgG into the chromatographic column. Again pour PBS buffer into the column.
- After that collect the solution into a sterile centrifuge tube labeled purified IgG.
- Take a cuvette labeled Blank (B) and add 1000 µl of PBS buffer into the cuvette. Similarly add 1000 µl of PBS buffer into another cuvette labeled B.
- Keep the blank cuvettes on the Spectrophometer and press “Enter” button to start the process. The graph in the monitor indicates the baseline.
- Add 1000 µl of purified IgG into a cuvette labeled test (T).
- Place the cuvette with purified IgG (T) into the spectrophotometer and press “Enter” button to start the process.
- Observe the resultant graph on the monitor.
- Transfer 5 ml of 50 % glycerol into a cryovial. Pipette 5 ml of purified IgG and add it into the cryovial containing 50 % glycerol.
- Wrap the cryovial with parafilm. Keep it on a cryobox and store it in the -20oC freezer for future purposes.
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