The In-Cell Western (ICW) Assay is a quantitative immunofluorescence assay performed in microplates (96- or 384-well format) that combines the specificity of Western blotting with the reproducibility and throughput of ELISA.
In-Cell Western assays are also called cytoblots, cell-based ELISA, In-Cell ELISA (ICE), and FACE (Fast Activated Cell-based ELISA). With In-Cell Western assays, you can:
In-Cell Westerns have been used for analysis of:
The ICW Assay is based on standard immunofluorescent methods.
In-Cell Western assays provide greater reproducibility and precision than Western blots. ICW assays exhibit the following characteristics:
Z′ factor is a statistic that measures the reproducibility of an assay for high-throughput screening. Z′ is calculated by running a large number of positive and negative controls, and determining how much separation there is between positive and negative controls. If they overlap, or nearly overlap (Z′ < 0.5), due to large amounts of variation within the controls, the assay is useless for screening. An excellent assay exhibits good separation between positive and negative controls (Z′ > 0.5).2, 4, 5
A 2010 study compared ICW Assays and Western blotting (WB) for measurement of phosphorylated myosin regulatory light chain (PMLC20).1 Primary cultures of uterine myocytes stimulated with oxytocin were used to assess specificity, sensitivity, and precision of the two methods for phospho-analysis.
In-Cell Western assay and Western blot analysis yielded very similar results (Fig. 3). ICW assays offered superior precision, reduced variability, and smaller CVs.
In-Cell Western assay results correlate well with immunoblot results and other assays.
Compared to Western blotting, ICW assays display:
GPCR activity assays primarily monitor upstream signaling events, such as accumulation of cyclic AMP (cAMP). Downstream events, such as phosphorylation of cAMP response element binding protein (CREB), may also be useful readouts.3, 6
The In-Cell Western method is commonly used to assess IC50 and has been shown to produce comparable IC50 results to other assays, such as radioligand binding affinities and cAMP accumulation assays.3
|Method of IC50 Determination||Result|
|Radioligand binding assay||8.40±0.05|
Normalization makes In-Cell Western analysis more precise by correcting for well-to-well variation in cell number.
Choose the normalization approach that best suits your experiment.
To simultaneously detect two target proteins in the ICW assay, you must:
A second protein target (such as actin, tubulin, COXIV, or GAPDH) can be used for normalization. Abundance of the normalization target must be unaffected by the cell treatments used.
It may be possible to use the target protein as its own internal control for detection of a phospho-protein.
Cell number normalization is a fast and inexpensive approach, because no additional antibodies are required. Options include CellTag 700 staining and cell labeling with reactive dye.
CellTag 700 Stain is a near-infrared fluorescent cell stain that provides accurate information to cell number for In-Cell Western applications. The stain accumulates in both the nucleus and the cytoplasm of permeabilized cells and provides linear fluorescent signal across a wide range of cell types and cell membranes. This method is fast and easy as staining is combined with secondary antibody incubation.
Using this method, cells are covalently labeled using cellular lysine residues on cellular proteins with IRDye 800CW or IRDye 700DX reactive dyes.7 Cell labeling is an inexpensive method with high sensitivity and a wide linear range1 (~200 to 200,000 cells/well) and results are unaffected by changes in nuclear DNA.
See some published examples of In-Cell Western AssaysView publications