electrophoresis to a nitrocellulose membrane, the protein was visualized, for loading

comparison, with fast green stain (50% methanol, 10% glacial acetic acid, 0.01% fast

green) by incubation at room temperature with gentle shaking on a Bellco Biotechnology

Orbital Shaker for 15 minutes followed by three 2-5 minute (depending on strength of

stain) destain washes (50% methanol, 10% glacial acetic acid). The fast green stain was

recycled for repetitive use and the destain was discarded. The membrane was washed

three times for 5 minutes with tris-buffered saline (TBS) (10 mM tris-HC1, 150 mM

NaC1, pH7.2) supplemented with 0.1% polyoxyethylenesorbitan monolaurate (tween 20)

(TTBS) and then blocked for 1 hour at room temperature or overnight at 4 oC in TBS

supplemented with 5% nonfat dry milk (NFDM). The primary antibody incubation was

performed with the anti-b subunit antibody (1:40) in TTB S supplemented with 2% B SA

for 1 hour followed by three 5 minute washes with TTBS. The anti-b antibody was

restored at -20 oC and thawed for reuse up to 3-5 times. Finally, secondary antibody

incubation was performed with horseradish peroxidase-linked donkey anti-rabbit

antibody (1:50,000 in TTBS-B SA), washed 4 times in TTB S (two 5 minute washes, two

10 minute washes) and then the antibody was detected by enhanced chemiluminescence

(ECL). Signals were visualized on high performance chemiluminescence film using a

Kodak X-Omat. Typical exposure times for the anti-b antibody ranged from 1-5 minutes

for strong signals and 30-60 minutes for weaker signals.

 Anti-HA antibody. The HA-epitope tag antibody was performed essentially as

described by the manufacturer followed by a secondary antibody incubation with

horseradish peroxidase-linked anti-mouse (from sheep) antibody (1:10,000). Several

initial attempts to visualize the HA-epitope tagged b subunit on a nitrocellulose