5%
milk blocked nitrocellulose Western blots of 293 cell
lysate were probed with Rabbit-anti-Human JNK1 (Santa
Cruz, sc-747) at 1:200 dilution, HRP-Goat-anti-Rabbit (Amersham)
at 1:10,000 dilution. The signal was detected using ECL
substrate (Panel A). Blots were stripped with either 2ml
of OneMinute®
Stripping Buffer (30
sec, RT) for 10 times or 10ml of competitor stripping
buffer (15min, 50℃) for 1 time. OneMinute®
Buffer treated blot was washed 3x 10 sec with TBST, and
directly re-probed without re-blocking. Competitor
stripping buffer treated blot was washed 3x 10min,
re-blocked with 5% milk for 1hour, and washed 3x 15min
with TBST. Blots were re-probed with the same primary and
secondary antibodies and detected with ECL substrate
(Panel B). A fresh identical blot was probed as a control
(left in Panel B) to evaluate the loss of JNK on
membranes.
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