The In-Cell Western™ Assay is an immunocytochemical assay that uses near-infrared fluorescence to detect and quantify proteins in fixed cells. Detecting proteins in their cellular context increases quantification precision. Proteins in fixed, cultured cells are detected directly in microplates, which yields higher throughput compared to Western blotting and eliminates typical Western blotting steps such as cell lysate preparation, electrophoresis, and membrane transfer. Using the In-Cell Western Assay kits, the cost per well for secondary screening is reduced to a fraction of the cost of typical screening methods. Watch an introductory webinar to In-Cell Western Assays.
The CellTag™ 700 Stain ICW Kits provide antibodies, blocking buffer, and CellTag 700 Stain to normalize well-to-well variations in cell number for forty 96-well plates or ten 384-well plates. Using protein stains reduces the cost per assay compared to performing the assay using two secondary antibodies. Any potential interference caused by using two antibodies is also eliminated.
Other In-Cell Western Assay Protocols are available on our Technical Resources Library. Visit our In-Cell Western Application Pages for more examples of how researchers have used these immunocytochemical assays.
Updated February 18, 2015.
AGAIN with the quantitative cell signaling! YES! because it is so versatile!! I am sure you will find that this will become a valuable technique to use in your research.
This quantitative immunofluorescent assay – the one that we call an In-Cell Western™ (ICW) Assay – can be used to study a variety of mechanisms. Here is an example of an ICW used to study apoptosis.
As you may already know, there are two major apoptosis signaling pathways: the death receptor (extrinsic) pathway and the mitochondrial (intrinsic) pathway. Under most circumstances, activation of either pathway leads to proteolytic cleavage and activation of caspases, a family of cysteine proteases that act as common death effector molecules. The In-Cell Western Assay is a very helpful research tool for scientists who are quantifying cell signaling.
Figure 1. 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).
Here is our complete apoptosis assay example protocol of the HeLa cellular response to anisomycin treatment (detailing the seeding, induction, and detection).