Hi Anette,

We perform all PEG extracts at 4 degrees, centrifugation needs at l;east 5000g, otherwise the yield is too less. The last step PEG with ice-cold ethanol needs 10000g aand 4 degrees, than teh yield is enough. We donot have experience with lower centifugation speeds not with temperature at ambient temperature.

Kind regards,

Anneke Geurts

I agree that t the centrifugation of PEG extracts shall follow the suggestion from Anneke Geurts-Moespot ·.

We prefer amoniumsulphate methods。 We compared the two kinds of methods: PEG and amoniumsulphate methods. We prefer the amoniumsulphate methods due to production of a high-quality IgY antibodies.

Thank you for your comments.

I have only briefly tried ammoniumsulphate precipitation. Both by adding saturated solution to a final concentration of 80% and by adding ammoniumsulphate in salt form. In both cases I found it a bit difficult to set the pH after addition of ammoniumsulphate. How do you do Ellen? Do you add solution or salt?

Dear Anette: Sorry, I am in meeting about 5 days, I will give you some suggestion latter. Ellen

Thanks

For your reference, we used two ways for isolation of the crude anti-TK1 IgY

The eggshell was washed with 70% ethanol and the egg yolk was separated from the egg white. The egg yolk was diluted with 5–10-fold volumes of deionized water under stirring. The crude solution was adjusted to pH 5.1, stored at 4 oC for 16–20 h and then centrifuged (15,000 g, 30 min). Subsequently, the supernatant solution was filtered throughout a 0.22-Am membrane filtration.

1. Extraction of the crude solution using the freeze-dried: The supernatant solution was lyophilized (See: Journal of Immunological Methods 277 (2003) 157– 169. Qimin He et al.,(Qimin He is Ellen He)

2. Extraction of the crude solution using the ammonium sulfate precipitation

The first ammonium sulfate precipitation: 1) Ammonium sulfate was added into the above the supernatant solution (40% ammonium sulphate solid)， stirring slowly until to dissolve, overnight at 4Co. 2) To discard the supernatant, centrifuge at 10 Co, 10000 g. 30 min. 3) The protein precipitation was dissolved with ddH2O (1:4).

Secondary ammonium sulfate precipitation: 1) Ammonium sulfate was added into the above the supernatant solution (23% ammonium sulphate solid)，stirring slowly until to dissolve, overnight at 4Co. 2) To discard the supernatant, centrifuge at 10Co, 10000 g. 30 min. 3) The secondary precipitation was dissolved with ddH2O (1:4), slowly adjusting the pH to 8.0 with 0.5M NaOH, and then adding 0.05% NaN3 before Affinity-purification column.

I hope that it can help you. Ellen

Is there not a risk of getting local spots of extremly high pH when adding solid ammonium sulphate? I don't want to denature the IgYs.

Thses is optimal pH of 5.1-8.0 and did not denature the IgYs