Azure Biosystems
In-cell Westerns (ICW) are an immunocytochemistry method that combines the specificity of Western blotting with the high-throughput of ELISA. In-cell Westerns are also called cell-based ELISA, cytoblots, or in-cell ELISAs (ICE).
The in-cell Western assay allows you to quantify proteins in cultured cells in situ, to assess the effects of drugs, or other interventions, on protein levels without further manipulation of the cells. ICWs provide an excellent, high-throughput way to examine how growth conditions, drugs, or other interventions change protein levels in cultured cells. An example of an ICW is shown below in Figure 1.
Biocompare BenchTip Article: How In-Cell Westerns Can Advance Your Research
Traditional Western blotting can be time consuming because protein samples must be extracted and the 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 compared to a traditional Western, which somewhat increases throughput. 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.
Figure 2 from BiteSize Bio provides a graphical comparison of in-cell Westerns, traditional Western blots, and ELISAs.
Read more: When to use ELISA vs Western blot
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. This process is shown in the Figure 3.
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.
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 in-cell Westerns 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 include those related to cell signaling, virology, and high-throughput drug screening.
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.
In addition to anti-viral agents, in-cell Westerns may also be used for high-throughput screening of drugs for activity against diseases, 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. An example of a drug screen using an in-cell Western is shown in Figure 4.
Performing in-cell Western blotting on 96-well plates allows for accurate measurement of intracellular protein expression in situ. It provides a high throughput methodology to assess multiple stimulations, end-points, proteins of interest and replicates on a 96-well plate.
Learn more about the ICW image above by reading this Customer Spotlight article: How the fluorescent intensity of proteins from in-cell Westerns are used to determine protein regulation in chondrocytes
By using NIR antibodies and a laser scanner like the Sapphire FL, the potential for in-well multiplex analysis exists, and offers further improvements to throughput.
The Sapphire FL is capable of in-cell westerns due to its sensitivity and speed. With four fluorescent channels (including two NIR channels), the Sapphire FL facilitates multiplex detection of blots and of 96-well plates with the AzureCyto In-cell Western Kit (Figure 5) like a pro. Learn more about the breadth of applications the Sapphire FL is capable of and schedule a free demo by clicking here.
The reagents you need for in-cell Westerns include: permeabilization solution, blocking solution, total stain, and both primary and secondary antibodies. For your convenience, these reagents are included in the AzureCyto In-Cell Western Kit from Azure.
This kit contains all reagents necessary for staining of whole cells for total cell normalization and simultaneous detection of two biomarkers and is enough to perform five 96-well in-cell Western assays.
From in-cell Western blotting to multiplex fluorescent Western blotting, whatever your Western blot needs are, we can help. If you’re still unsure of which system you need for the in-cell Western application, one of our experts is available to help you choose the right option. Fill out the form on this page and choose the applications your lab uses. We’ll contact you to match you up with the best fit.
Enables true multiplex detection by using NIR 700 and NIR 800 channels for target detection
Easy quantitative analysis of in-cell Western blots, Western blots, bacterial colonies, and more
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.
Looking for a full list of applications?
Both the Azure 300 and Sapphire Biomolecular Imager, along with fluorescent blocking buffer and secondary antibodies from Azure Biosystems, were used in a recent publication revealing anti-inflammatory activity of a mosquito saliva
Western blotting vs. In-cell Western blotting Western blotting was first described by three groups in 1979. Since then numerous refinements to the basic technique, reagents
Western blots, step aside… Despite not having quite such a catchy name as Western blotting, in-cell Western blotting (ICW) is a simple technique that offers many advantages and
