Protein Protocols Protein Protocols

1010 Bird and Thorpe
4. Centrifuge.
5. Anhydrous sodium sulfate.
6. Saturated sodium sulfate solution: 36% (w/v) in water.
3. Methods
Break the eggshell and carefully separate the egg yolk from the white to minimize
contamination of the yolk with egg white proteins. Place the yolk on a piece of filter
paper in which a small hole has been cut. Position the yolk above the hole and pierce
the yolk membrane with a needle and collect the yolk in a suitable container (e.g.,
50-mL centrifuge tube). The yolk membrane and any remaining egg white will stick to
the filter paper. Approximately 10 mL of yolk is obtained from an average sized egg.
1. Dilute the egg yolk with 4 vol of TBS.
2. Centrifuge at 2000–3000g for 20 min at room temperature with the brake off.
3. Remove the supernatant, and discard the membrane pellet and add 120 µL of dextran
sulfate solution/mL of supernatant, mix well, and incubate at room temperature for 30 min.
4. Add 50 µL of 1 M CaCl2/mL and mix well. Incubate for a further 30 min at room temperature.
5. Centrifuge at 2000–3000g for 30 min and collect the supernatant, which should be clear;
if not, repeat steps 3 and 4 using half the amounts of dextran sulfate and calcium chloride.
The pellet can be washed once at this stage with 50 mL of TBS, and the supernatant
combined with the first dextran sulfate supernatant obtained.
6. Adjust the volume of the pooled supernatants to 100 mL with TBS. Stir the supernatant
and slowly add 20 g of sodium sulfate until completely dissolved and leave to stand for
30 min at room temperature.
7. Centrifuge for 20 min at 2000–3000g and discard the supernatant.
8. Redissolve the precipitate in 10 mL TBS and centrifuge for 20 min at 2000–3000g. Collect
the supernatant and discard the pellet.
9. Stir the supernatant and slowly add 8 mL of 36% (w/v) sodium sulfate solution and leave
to stand for 30 min at room temperature.
10. Centrifuge at 2000–3000g for 20 min, discard the supernatant, and redissolve the pellet in
5 mL of TBS. Dialyze and filter the purified immunoglobulin.
4. Notes
1. Eggs may be stored at 4°C until several have accumulated and then processed together.
Alternatively separated egg yolk can be diluted with buffer containing preservative and
stored at 4°C prior to purification.
2. 36% (w/v) Sodium sulfate is a supersaturated solution and requires heating to fully dissolve,
after which the solution should be stored at 30–40°C before use. The entire procedure
for IgG precipitation with sodium sulfate should be carried out at 20–25°C; otherwise
the sodium sulfate may precipitate.
3. Purified chicken IgG can sometimes aggregate after freezing and thawing; therefore storing
sterile solutions of purified chicken IgG at 4°C may be advisable; alternatively they
can be lyophilized for longer term storage.
4. The chicken IgG heavy chain has a different mobility from mammalian IgG, which can be
demonstrated by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) (Fig. 1).