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.