| 5% milk
nitrocellulose Western blots of HeLa cell lysate proteins were probed with
Mouse-anti-Human RIPK1 (protein
1, BD, 610458) at 1:200 dilution, HRP- Goat-anti-Mouse (Amersham) at
1:2,000 dilution. The signal was detected using ECL(Panel A). Blots were stripped with
2ml of OneMinute®
Stripping Buffer (30sec,
RT),
or 10ml of competitor stripping buffer (P's R product) for
either 30 sec or 15min at RT, and then incubate to ECL to
evaluate the stripping efficiency of secondary antibody (Panel B). 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, blocked with 5% milk for 1hour, and washed 3x 15min with TBST. Blots were re-probed with
Mouse-anti-Human IKKgamma (protein
2, BD, 559675) at 1:200 dilution,
HRP-Goat-anti-Mouse (Amersham) at 1:2,000 dilution and detected with ECL(Panel
C). The removal of the primary
antibody against Protein 1 was also evaluated by using
same secondary antibodies (Anti-Mouse). 15min stripping
with competitor P's R product completely removed
secondary antibody (right lane in Panel B) but failed to
completely remove primary antibody (right lane in Panel
C). |
