In-cell Westerns

What is an in-cell Western?

Cell-based ELISA, also called in-cell ELISA (ICE) or in-cell Western (ICW), is an immunocytochemistry method that combines the specificity of Western blotting with the high-throughput of ELISA. An example of an ICW is shown below in Figure 1. The approach allows you to quantify proteins in cultured cells in situ, assessing the effects of drugs or other interventions on protein levels without further manipulation of the cells.

In-cell Western taken from Azure Sapphire Biomolecular Imager
Figure 1. HeLa cells were serially diluted and seeded into a 96-well plate, cultured, fixed and permeabilized, then probed for Tubulin with AzureSpectra 550 (520 channel, green), beta-Actin with AzureSpectra 800 (785 channel, blue) and RedDot™1 Nuclear Stain as a normalization control (785 channel, red).

With in-cell Westerns you're able to:

  • Detect proteins in fixed cultured cells using target-specific primary antibodies and fluorescent secondary antibodies
  • Quantify up to 4 targets per well
  • Quickly, accurately measure relative protein levels in many samples
  • Detect proteins in situ in a relevant cellular context

Advantages of using in-cell Westerns

Traditional Western blotting can be time consuming. This is because protein samples must be extracted and electrophoresis and transfer steps must be conducted before antibody binding and detection can be carried out. When size information isn’t required, dot or slot blotting can save time by bypassing the electrophoresis step. With the addition of a special apparatus, many more samples can be analyzed on one blot than on a traditional Western, increasing throughput somewhat. However, before dot or slot blotting, protein extracts must be prepared from each cell type or condition being assessed.

ELISA (enzyme-linked immunoassay) provides a high-throughput approach to quantifying the presence of a protein or other antigen in a sample. ELISA relies on antibodies specific to the protein or antigen of interest coating the bottom of the wells of a 96- or 384-well plate to quickly capture the target when samples are added to the wells. However, an ELISA also requires extracting proteins from the cells of interest which increases the time to obtain results, and also introduces the potential to lose or alter the antigen of interest during the cell harvesting, lysing, and other extraction steps.


Visual demonstration of the in-cell western protocol

ICWs turn ELISA on its head, with cells (target) bound to the bottom of the wells of a 96- or 384-well plate rather than an antibody. The cells are fixed and permeabilized in the culture dish where they were grown, and then the target protein is detected much like in a traditional Western blot. The cells are blocked to prevent nonspecific binding, incubated with the primary antibody, washed and then incubated with a labeled, secondary antibody.

For ICWs, the secondary antibody is usually labeled with a near-infrared (NIR) fluorophore, which is less subject to interference from auto-fluorescence or from noise from the plastic of the tissue culture dish. A nuclear stain can be used to normalize the signal from the Western to the number of cells in the well. In-cell Westerns provide an excellent, high-throughput way to examine how growth conditions, drugs, or other interventions change protein levels in cultured cells.

What questions can you answer with in-cell Westerns?

With ICWs, you can answer questions about protein levels inside and on the surface of cultured cells, and how those protein levels change in response to cell treatments. Because they are high-throughput, streamlined, and quantitative assays, they can be used to answer questions that require multiple replicates or testing numerous conditions in an efficient, cost-effective manner. Some examples of the types of questions to which the in-cell Western approach has been applied are discussed below.

Cell Signaling

In-cell Westerns are frequently used in studies of cell signaling to answer questions about protein phosphorylation levels, or provide insight into the activities of kinase signaling pathways such as the MAP kinase cascade. In addition, they can be used to study the abundance of cell surface proteins, ligand binding to cell surface proteins, and cell surface receptor internalization and recycling. These are all important to understanding cell signaling.


In-cell Westerns have multiple applications in the field of virology. This application can be used to assess viral titers in order to answer questions such as how well different viral strains replicate in different types of host cells. In-cell Westerns may also be applied to high-throughput screening of potential antiviral agents, to identify drugs that block or interfere with viral infection.

High-throughput screening of drugs

In addition to anti-viral agents, in-cell Westerns may also be used for high-throughput screening of drugs for activity against diseases, including cancers, or bacterial infections. In-cell Western assays can assess small compounds, siRNAs, or other potential therapeutics for anti-disease activity by quantifying proteins that serve as a biomarkers of disease progression.

What to use to image in-cell Westerns

Ready to start with in-cell Westerns? The Azure Sapphire Biomolecular Imager is an imager capable of in-cell westerns due to its sensitivity and speed. With four fluorescent channels including two NIR channels, the Sapphire facilitates multiplex detection like a pro.

Related Products
Azure Sapphire Biomolecular Imager

Enables true multiplexed detection by using NIR 700 and NIR 800 channels for target detection 

Featured Publications

Authors from this paper measure the intensity of phospho-ERK1/2 and ERK1/2 proteins using in-cell Westerns in a 96-well plate using the Sapphire.

Additional Resources

Looking for a full list of applications?

Related blog posts about in-cell Westerns…
In-cell Western taken from Azure Sapphire Biomolecular Imager
In-cell Western

History Behind In-cell Westerns

Western blotting was first described by three groups in 1979. Since then numerous refinements to the basic technique, reagents used and imaging technologies have massively

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