Separate the egg yolk from the white and place the yolk into a 50ml graduated cylinder. Record the volume as "x" ml.
Place the egg yolk into a plastic centrifuge bottle and add to it 4x ml of Buffer and (3.5(5x))/100 g of PEG 8000. Shake gently to dissolve PEG.
Centrifuge at 5000g (5500 rotations per minute (rpm) JA-10 rotor in the Beckman refrigerated centrifuge for 20 minutes.
Pour the supernatant into a 250ml graduated cylinder and record the volume as "y". Discard the precipitate.
Pour the supernatant into a clean centrifuge bottle and add to it (8.5y)/100 g of PEG 8000. Let stand at room temperature for 10 minutes to allow the precipitate to form.
Centrifuge at 5000g (5500 rpm) in the Beckman centrifuge for 25 minutes.
Discard the supernatant; drain excess onto a paper towel. Add 2.5x ml of Buffer to the precipitate and dissolve by stirring or vortexing gently. Transfer the liquid to a fresh centrifuge bottle.
Add (12(2.4x))/100 g of PEG 8000 and swirl to dissolve. Let stand at room temperature for 10 minutes to allow precipitate to form.
Spin 5000g (5500 rpm) in the Beckman refrigerated centrifugefor 25 minutes.
Discard the supernatant and drain excess on a paper towel. Add (1/4)x ml of Buffer to the precipitate and vortex to dissolve.
Place on ice for 10 minutes.
Add (1/4)x ml of 50% ethanol (precooled to -20°C) to the tube on ice and vortex to mix. Transfer to a precooled Flip cap tube.
Centrifuge 10,000g for 25 minutes.
Discard the supernatant as before and dissolve the precipitate in (1/4)x ml of Buffer.
Place the dissolved precipitate into dialysis membrane using the appropriate clips and record the number on the clip corresponding to the particular egg. Place the dialyse membranes into a flask containing 50(1/4)x ml of Buffer per egg.
Add a magnetic stir bar to the flask and place on a stir plate in the cold room. Dialyse with stirring for 24 hours. Label the flask with the solution name and your name and the date.
After dialysis remove the solution from the membrane and place into a 15ml plastic tube. Label and date each tube and add 0.1% Na azide as a preservative. Store at 4°C in the cold room.
Buffer: 0.1M phosphate buffer, pH 7.6
To make: add 0.1M KH2PO4 to 0.1M K2HPO4 until pH is 7.6.
