In-Cell Western™ Assay

The In-Cell Western (ICW) Assay is a quantitative immunofluorescence assay performed in microplates (optimized for 96- or 384-well format) that combines the specificity of Western blotting with the replicability 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:

Detect proteins in fixed and permeabilized cultured cells using target-specific primary antibodies and IRDye^® secondary antibodies
Quantify two targets at 700 nm and 800 nm, using spectrally-distinct infrared dye conjugates
Quickly, accurately measure relative protein levels in many samples
Detect proteins in situ in a relevant cellular context

**Figure 1.** Analysis of Akt phosphorylation in MCF-1 breast cancer cells in the presence of increasing concentrations of IGF-1. A) In-Cell Western plate image. B) pAkt intensity in 800 channel was normalized to total Akt intensity in 700 channel.

In-Cell Westerns have been used for analysis of:

The In-Cell Western Assay is a valuable tool for quickly characterizing a broad range of cell signaling parameters in the development of targeted therapeutics. Consider DigiWest^® Protein Profiling Services to give you a head start in identifying potential therapeutic targets or the functional effects of a therapeutic. Then investigate further using and In-Cell Western Assay.

How Does the In-Cell Western Assay Work?

The ICW Assay is based on standard immunofluorescent methods.

Culture cells in microplates (adherent or suspension cells).
Treat cells.
Fix and permeabilize.
Add the blocking buffer.
Stain with primary antibodies - 1 or 2 protein targets per well.
Stain with IRDye secondary antibody conjugates.
Image microplate.
Quantify relative protein levels.
Normalize with cell stain, such as CellTag™ 700 Stain, or primary antibody detection to correct for well-to-well variation

See an example of a typical In-Cell Western workflow.

In-Cell Western Assays are Replicable and Precise

In-Cell Western Assays provide greater replicability and precision than Western blots. ICW assays exhibit the following characteristics:

Shorter protocol
Significantly smaller standard deviations
Replicate measurements with very low coefficients of variation (CVs) (Fig. 2 and 3)
Easily run many replicates to increase accuracy (Fig. 4)
Characterize a broad range of cell signaling parameters
Very similar profiles of signal increase and decrease when compared to Westerns
Produces excellent Z′ factors with optimized conditions and experimental design

Z′ factor is a measure of statistical effect size and can be used to assess if a response to an assay requires further investigation. Z′ factor can provide some indication of the replicability of an assay. 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.

**Figure 2. Intra-plate variability of ICW is very low.** HeLa cells were treated with 10 μm chloramphenicol (CAM) or vehicle (DMSO) for 7 days. Levels of OXPHOS Complex IV (COX-1, MS404; Mitosciences) were analyzed by In-Cell Western. Statistics are shown for background (no primary antibody; n=16), DMSO, and CAM treatments (n=40 for each; CVs = 6% and 4%, respectively).

**Figure 3. Intra-assay variability of Western blots and ICW assays.** A) GAPDH and PMLC20 were detected on duplicate Western blots (13 replicates per blot; not shown). Band intensities are expressed as a proportion of the mean of the thirteen samples on each blot (blot 1 in blue, blot 2 in red). CV was 0.27. B) Signal intensities from ICW wells are plotted as a proportion of the mean values for each of two plates (plate 1 in blue, plate 2 in red). CV ranged from 0.08 to 0.16. Aguilar, HN et al. PLoS ONE 5(4): e9965 (2010). doi:10.1371/journal.pone.00099

**Figure 4.** Replicability of ICW for siRNA screening. A pilot small molecule siRNA screen was performed for inhibitors of mTORC1 signaling. A library of ~2500 known bioactive compounds was used. Compounds with average Z-score < -2 are considered hits (red circles). Known inhibitors of mTORC1 signaling found in the library are shown in green. Plate-to-plate consistency is shown for a representative plate from the library. Adapted with permission from Hoffman, GR et al.²

Correlation of In-Cell Western Assays with Other Assays

In-Cell Western Assay results correlate well with immunoblot results and other assays.

Correlation of In-Cell Western Assays with Western Blots

Compared to Western blotting, ICW assays display:

Very similar profiles of signal increase and decrease, and similar IC₅₀ values and sensitivity
Biologically and physiologically validated data output¹
Improved replicability and precision¹

Significantly smaller standard deviations
Replicate measurements with very low CVs

Higher throughput, to easily process many samples or replicates in parallel

IC50 Values from ICW Assays Correlate Well with Published Values from Other Assays

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
In-Cell Western	8.72±0.13
cAMP	8.72±0.07
Radioligand binding assay	8.40±0.05

Table 1. pKi, the negative log of the inhibition constant of a drug; pKB, the negative log of the dissociation equilibrium constant for a competitive antagonist; ap, apparent; CREB, cAMP response element binding protein. Selkirk JV et al. J. Biomol. Screen. 11: 351 (2006).

Typical In-Cell Western Assay Workflow

Normalization of In-Cell Western Assays

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.

Normalize to an Internal Control Protein

To simultaneously detect two target proteins in the ICW assay, you must:

Use two primary antibodies that differ in either host species or subclass/isotype
Use secondary antibodies labeled with different IRDye fluorophores (IRDye 680RD for 700 nm channel and IRDye 800CW for 800 nm channel)

Housekeeping Protein

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, which must be validated before the assay is performed.

Combined Detection of Pan Protein and Phospho-protein Levels

It may be possible to use the target protein as its own internal control for detection of a phospho-protein.

Total levels of the target protein and extent of phosphorylation of the target are measured simultaneously.
Combine the phospho-antibody with a "total protein" antibody that recognizes the target protein regardless of its phosphorylation status. For example, use mouse anti-total Akt and rabbit anti-phospho-Akt.
This method also corrects for changes in target protein abundance that may be caused by cell treatments.
When using two antibodies against the same target, antibody interference may occur (one antibody blocks binding of the other antibody). Controls to rule out antibody inference are very important.

Normalization to Cell Number

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 Staining

CellTag 700 Stain is a near-infrared fluorescent cell stain that provides accurate estimation of 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.

Cell Labeling with Reactive Dye

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 range (~200 to 200,000 cells/well), and results are unaffected by changes in nuclear DNA.

See some published examples of In-Cell Western Assays

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