IRDye® 800CW Applications
Multiplex Western Blot Detection
Figure 1. Example of a Multiplex Western Blot. Lysates of EGF-treated A431 cells were separated and transferred to nitrocellulose. The blot was probed with anti-ERK and anti-phospho-ERK primary antibodies, and then detected with IRDye 680LT and IRDye 800CW secondary antibodies. Blot was imaged with Odyssey Fc System for 2 min. This phospho-ERK antibody cross-reacts with phospho-EGFR (upper green band).
In-Cell Western™ Assay
Figure 2. Time course of caspase-3 activation in S2 cells. (A-C) In-Cell Western analysis of S2 cells treated with Actinomycin D (Act D) to induce apoptosis. Each time point was measured in triplicate and stained for anti-active-caspase-3 (A; green) and f-actin (B; red, stained with near-infrared fluorescent phalloidin). Panel C shows merged pseudocolor images. (D) Active-caspase-3 protein levels from (A) were quantified and normalized to f-actin levels in (B) for each time point. The active caspase-3:f-actin ratio at 0min Actinomycin D exposure was designated as 1, and all other ratios are shown relative to this value. Error bars represent the standard error of each independent measurement. Exposure of S2 cells to Actinomycin D increased the relative levels of active caspase-3 over time.
Reprinted with permission from Bond, D.et al. Biol Proced Online. 10(1):20-28(2008).