Alkaline phosphatase conjugated goat anti rabbit antibody

Samples prepared by SDS-PAGE as described above were transferred to nitrocellulose membranes at 30 V for 1 hour. Membranes were blocked with 5% (w/v) milk in TBS-Tween (150 mM NaCl, 10 mM Tris HCl, 0.1% (v/v) Tween-20, pH 7.5) for 1 hour at RT. Membranes were then incubated with 0.5 μg/mL rabbit polyclonal anti-TMV antibody (custom-made, Pacific Immunology) or rabbit polyclonal anti-albumin antibody (NBP1-32458, Novus Biologicals) in 5% (w/v) milk in TBS-Tween overnight at 4°C. Membranes were then washed three times for 10 min each in TBS-Tween, followed by incubation with 1 μg/mL alkaline phosphatase-conjugated goat anti-rabbit antibody (G21079, Thermo Fisher Scientific) in 5% (w/v) milk in TBS-Tween for 1 hour at RT, followed by three washes for 10 min each in TBS-Tween and one 5 min wash in dH₂O. Antibody binding was visualized using Novex AP Chromogenic Substrate (BCIP/NBT; Invitrogen).