Secondary anti-rabbit HRP (GE Healthcare) was used at 1 5000 dilution for 1 h at RT and the blots were then washed as above.
The blots were then washed in 1x TBST four times for 10 min.
The blots were then washed 3× for 10 min each time, with TBS-T.
After overnight incubation, the blots were then washed 3× (each 10 min) with TBS-T and incubated at room temperature for 1 h with horseradish peroxidase conjugated anti-rabbit IgG secondary antibody (1 5000) diluted in TBS-T.
The blots were then washed with TBS, and blocked with TBST.
The blots were then washed in 1x TBS-Tween for 1 hour and films exposed using ECL solutions.
The blots were then washed with TBST and incubated with alkaline phosphatase-conjugated secondary antibody diluted 1∶500 in the blocking buffer at RT for 1 h.
Blots were then washed with TBST and incubated with the primary Ab diluted in the appropriate Ab-specific blocking solution suggested by the manufacturer.
Blots were then washed three times with TBS-T and incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (Molecular Probes, Eugene, OR) for 1 h at room temperature.
The blots were then washed three times with PBS-Tween 20 before the reaction with enhanced-chemiluminescence, Western blotting detection reagents (Amersham).
The blots were then washed, exposed to HRP-conjugated secondary antibodies (1∶5,000 dilution) for 1 h, and the antigen-antibody complex was detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ, USA).
