membrane failed to give a clean signal. It was essential to enhance the sensitivity of the

antibody by transfer onto a PVDF membrane for a higher signal to noise ratio. Before

electrotransfer, it was crucial to prepare the membrane by immersion in 100% methanol

for 15 seconds ddH20 for 2 minutes and then transfer buffer for 5 minutes. The wash

buffer consisted of a phosphate buffered saline (PBS) (1% NaC1, 0.025% KC1, 0.18%

Na2HPO4, 0.03% KH2PO4, pH 7.4) supplemented with 0. 1% tween 20 (PBST),

membranes were blocked in PBS with 5% NFDM, and the primary and secondary

antibody incubation was performed in PBST supplemented with 2.5% NFDM. Typical

exposure times for the anti-HA antibody were about I hour. Occasionally, a stronger

detection reagent, ECL Plus (Amersham Biosciences), was required for detection. ECL

Plus is an extremely sensitive detection system; therefore it was necessary to follow

protocol carefully, using optimal primary and secondary antibody conditions. The

detection reagents were brought to room temperature before opening and then solutions A

and B were mixed in a 40:1 ration. The final volume required was 0.1 mL per cm2 Of

membrane. Excess wash buffer was allowed to drip off the membrane and then the

detection solution was pipetted onto the membrane and incubated at room temperature for

5 minutes. Finally, it was critical to drain off excess detection reagent by touching the

corner of the membrane onto a kimwipe before exposure to film.

 Anti-V5 antibody. The V5-epitope tag antibody incubation was performed as

described by the manufacturer followed by secondary antibody incubation with

horseradish peroxidase-linked sheep anti-mouse antibody (1:10,000). The wash buffer

consisted of TTBS, membranes were blocked in TBS with 5% NFDM, and the primary

and secondary antibody incubation was performed in TBS supplemented with 2.5%