Nitrocellulose n membrane

Samples of transgenic and UC plants’ leaf tissue were homogenized in 50 mM Tris-HCl buffer (pH 9.0). The extracts were centrifuged at 5,000 g for 20 min at 4 °C, and supernatants were collected and the protein samples (5 μg) were subjected to 15% SDS-PAGE^{63 (link)}. Subsequent to electrophoresis, the separated proteins were transferred onto nitrocellulose N-membrane (Amersham) by electroblotting^{64 (link)}. After protein transferring, the membrane was blocked by incubating in PBS solution containing 10% non-fat dried milk and 0.1% Tween 20 for 2 h at room temperature. Later, the membrane was probed with polyclonal rabbit anti-ASAL serum (1:10,000 dilution) as primary and goat anti-rabbit IgG horse-radish peroxidase conjugate (GENEi) as secondary antibody (1:10,000 dilution). The membrane was washed and treated with saturated benzidine solution containing 20% ammonium chloride and 0.1% H₂O₂.

Insects fed on transgenic rice lines and untransformed control plant were collected and homogenized in 50 mM Tris-HCl buffer (pH 9.0) to isolate total proteins of the insects. The extract was centrifuged at 5,000 g for 20 min at 4 °C, and the supernatant was collected and the protein samples (5 μg) were subjected to 15% SDS-PAGE^{63 (link)}. The separated proteins were transferred onto nitrocellulose N- membrane (Amersham) by electroblotting^{64 (link)}. After protein transfer, the membrane was blocked by incubating in PBS solution containing 10% non-fat dried milk and 0.1% Tween 20 for 2 h at room temperature. The membrane strips were probed with polyclonal rabbit anti-ASAL serum (1:10,000 dilution), followed by goat anti-rabbit IgG horse-radish peroxidase conjugate (GENEi) as a secondary antibody (1:10,000 dilution). Membrane strips were washed and treated with saturated benzidine solution containing 20% ammonium chloride and 0.1% H₂O₂.