- Phosphate buffer (100 mM sodium phosphate, pH 7.6).
- Storage phosphate buffer (100 mM sodium phosphate, pH 7.6 containing 60% glycerol)
- Distilled water
- Polyethylene glycol 6000 (PEG 6 kDa). Crush this with a mortar and pestle to speed up dissolution when performing fractional precipitation.
- Cotton wool
- Centrifuge tubes
- A big bag for disposal of waste
- Rotary (windmill) mixer
- Egg separator
- Pasteur pipettes [sterile, plastic]
- Measuring cylinder (25 ml, 250 ml)
- Small Erlenmeyer flasks [50 or 100 ml]
- Separate egg yolks from the egg whites using an egg separator and wash gently with distilled water using a Pasteur pipette.
- Puncture the yolk sac with a sterile pipette and measure the volume. Try to prevent the yolk sac from falling into the measuring cylinder. Record the yolk volume.
- Add two volumes of phosphate buffer and mix gently by inversion after sealing the cylinder with Parafilm.
- Add crushed PEG to 3.5% (m/w) and dissolve by gently stirring or by rotary inversion. After 10-15 minutes, you will notice that a yellow, oil substance has come out of solution and it will probably stick easily to the sides of the tube. The remaining supernatant fluid will appear slightly cloudy. Do not proceed further until you have noticed this.
- Pellet the yellow substance [this is the vitellin fraction, containing lipoproteins] by centrifugation (4420 x g, 30 min, RT).
- Moisten cotton wool with distilled water and place in funnel. Place funnel into Erlenmeyer flask.
- Pour off the supernatant and add it to the moisten cotton wool. This will trap the contaminating lipids. Measure and record the volume of the filtrate.
- PEG (8.5% [m/v]) is added to the clear filtrate to bring the final PEG concentration to 12% (as PEG was added to 3.5% in step 4). The solution is mixed until all the PEG has dissolve. Wait 15-20 min and then centrifuge (12000 x g, 10 min, RT).
- The pellet is dissolved in phosphate buffer, in a volume equal to that obtained after filtration through cotton wool.
- Add PEG to 12% (m/v), mix to dissolve and, after 15-20 min, centrifuge (12 000 x g, 10 min, RT).
- Discard the supernatant fluid, wash the pellet briefly with phosphate buffer and dissolve the pellet in 1/6th of the original egg yolk volume using phosphate buffer containing 60% glycerol. It is easiest to add the buffer to the pellet and leave overnight in the fridge. That way, the pellet will redissolve slowly. Aliquot the purified IgY into Eppendorf tubes and determine the concentration at 280 nm of a 1/50 dilution using an extinction coefficient of 1.36 [i.e., a 1 mg/ml solution of pure IgY will give an absorbance of 1.36 at 280 nm in a quartz cuvette with a path length of 1 cm].
- Store the purified IgY at –20°C until required.
- For western blotting and ELISA, use a starting concentration range of 5-20 μg/ml. For immunogold EM, use 10-50 μg/ml.