Immunotransfer & Immunoblotting
Immunotransfer was undertaken using an iblot system (Invitrogen) with nitrocellulose membranes according to manufacturer’s protocol. The nitrocellulose membrane was blocked with blocking buffer (5% Skim milk powder in Tris-buffered saline (TBS)) for 1 h. All incubations and washes were performed on a rotating shaker. The membrane was washed twice in TBS for 2 min, and then the primary antibody (Rabbit anti-TeNT IgG, ab53829, Abcam) diluted to 1:10,000 in blocking buffer was added for 2 h at room temperature (or overnight at 4ºC). The blot was washed twice in TBS for 5 min, and the secondary antibody (Goat anti-rabbit IgG conjugated with AP, ab6722, Abcam) diluted to 1:10,000 in blocking buffer was added and incubated at room temperature for up to 2 h. The blot was washed three times in TBS 0.05% tween for 5 min. To develop the AP conjugate, the blot was immersed in Western BlueTM (Promega) solution for 5-10 min. until the desired resolution was achieved. The blot was washed in dH2O to stop the reaction.