5% milk blocked PVDF Western blots of
HeLa cell lysate were probed with
Mouse-anti-Human RIPK1
(protein1, BD, 610458) at 1:200 dilution, HRP-Goat-anti-Mouse
(Amersham) at 1:2,000 dilution. The signal was detected using ECL substrate (Panel A). Blots were stripped with either
2ml of OneMinute®
Stripping Buffer(30sec,
RT) or 10ml of competitor stripping buffer
(P's R product, 30sec, RT) (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 then re-probed with Mouse-anti-Human IKKgamma
(protein2, BD, 559675) at 1:200 dilution, HRP-Goat-anti-Mouse
(Amersham) at 1:2,000 dilution and detected with ECL substrate (Panel C).
The removal of the primary
antibody against Protein 1 was also evaluated by using
same secondary antibodies (Anti-Mouse).
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