After washing the membranes with Tris-buffered saline-0.05% Tween 20 (washing buffer), target proteins were detected with alkaline phosphatase (AP -labeled ReserveAP Rabbit anti-goat immunoglobulin G (IgG) (KPL, Gaithersburg, MD, USA; 1 1,000 dilution in 1% PBS containing 0.05% Tween 20).
After four more washes with TBS-T buffer, immunoreactive proteins were detected with an enhanced chemiluminescence kit (Western Lightning™, Westernblot Chemiluminescence Reagent Plus, PerkinElmer Life Sciences, Waltham, MA) according to the manufacturer's instructions.
After washing the cells three times with PBA buffer, bound proteins were detected by anti-human TRAIL mAb MAB687 (2.5 μg/ml) and fluorescein isothiocyanate (FITC -labeled rabbit anti-mouse IgG Ab (1 : 200, Sigma-Aldrich), FITC -labeledhrabbitshing steps with PBA each.
The reaction was terminated by the addition of Laemmli sample buffer and phosphorylated proteins were detected by SDS-PAGE followed by autoradiography.
Following washing in 0.1% Tween TBS buffer, the immunolabeled proteins were detected by enhanced chemiluminescense reagents (Applygen, Beijing, China).
The beads were washed and boiled in the SDS loading buffer, and the precipitated proteins were detected by Western blotting.
The beads were washed extensively and boiled in SDS loading buffer, and the precipitated proteins were detected by Western blotting.
Proteins were detected by incubation with primary antibodies at appropriate dilutions in blocking buffer overnight at 4°C.
All proteins were detected by enhanced chemiluminescence.
Immunoreactive proteins were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech).
