Cut membrane, loading sample, blocking

1. According to the amount of sample to cut 8 cm × 6 cm nitrocellulose membrane. Gently draw on rectilinear reference lines per 1cm to separate the membrane into 48 grids, then marked with numbers.
2. Dilute the peptide into 5 ug/mL by 2 mL PBS (pH 7.4). Drop 2 uL the dilution slowly to the center of the grid . Adding sample order: the upper row is NP-pep , the nether row is P-pep . Each antigen should be loaded two grids. Place 30 min to dry.
3. According to the number to cut the membrane as a unit with two grids. Incubate the membrane in blocking buffer (5% non-fat milk in TBST (m/v)) for 1 hour at room temperature.

Antibody Dilution

1. The antibody should be diluted into 0.5 ug/mL by the blocking buffer.

Incubation Primary Antibody

1. Put the blocking membrane into the diluted antibody solution for 1 hour at room temperature.

Incubation secondary antibody

1. (HRP labeled goat anti rabbit Ig antibody)
2. Washing the membrane with TBST (pH 7.4) for 3-5 min.
3. Dilute the secondary antibody with the blocking buffer.
4. Incubation secondary antibody for 1 hour at room temperature.

Coloration and Developing

1. Washing the membrane with TBST (pH 7.4) for 3 - 5min.
2. Put the membrane into the substrate of HRP solution for coloration for 7 min.
3. Develop.