Western blot Protocol

1.  Block membrane with 5% non-fat milk in PBS-T for 1 hour at room temperature or longer at 4C.
2.  Incubate membrane with IgY antibodies at dilution of 1: 2,000 with 1% milk in PBS-T at R.T. for 1 h.
3.  Rinse 3 times with PBS-T, then wash membrane with PBS-T, 5 min each, total of 3 times.
4.  Incubate with 2nd antibody (goat-anti-IgY/Fc-HRP) at dilution 1: 1,000 for ECL (with 1% milk PBS-T) at R.T. for 1 h.
5.  Rinse 3 times with PBS-T, then wash with PBS-T, 5 min each with shaking, total of 3 times.
6.  Perform ECL detection of signal using Pierce ECL kit.