In-cell Westerns

Contents

What is an in-cell Western?

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.

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). The individual channels were scanned simultaneously then combined into a single composite image using the Sapphire Capture Software.

In-cell Westerns allow you to

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

Comparing In-cell Westerns to traditional Western blots and ELISAs

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

Venn diagram comparing in-cell Westerns, traditional Western blots, and ELISAs.
Figure 2. A Venn diagram comparing in-cell Westerns, traditional Western blots, and ELISAs.

In-cell Westerns vs. ELISAs

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.

Visual demonstration of the in-cell western protocol
Figure 3. Visual demonstration of the in-cell western protocol

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.

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 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.

How to image in-cell Westerns

Performing in-cell Western blotting on 96-well plates allows for accurate measurement of intracellular proteins expression in situ. It provides a high throughput methodology to assess multiple stimulations, end-points, proteins of interest and replicates on a single plate. 

In cell western screen for Type II Collagen imaged using Azure Sapphire Biomolecular Imager
Figure 4. In-cell Western scanned using the Sapphire provided by the Parreno Lab from the University of Delaware. Eleven drugs were screened for their ability to promote cartilage deposition in cells.

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

Imagers capable of imaging 96-well plate in-cell Westerns

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.

Sapphire FL biomolecular imager
The Sapphire FL is the ultimate biomolecular imager for flexibility. With customizable and user-changeable laser and filter modules, the Sapphire FL easily adapts to a lab’s changing needs and advancing research.

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.

1:2 serial dilution of HeLa cells seeded into a 96-well plate
Figure 5. 1:2 serial dilution of HeLa cells seeded into a 96-well plate. (A) A composite image of three channels imaged simultaneously on the Sapphire™ FL at 100-micron resolution. (B) hnRNP K visualized with the 685 Standard Optical Module. (C) GAPDH visualized with the 784 Standard Optical Module. (D) AzureCyto stain visualized with the 532 Standard Optical Module. All images were taken on a Sapphire™ FL Biomolecular Scanner.

Reagents needed for imaging in-cell Westerns

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.

how to use the azurecyto in-cell western reagent kit
Each AzureCyto In-cell Western Reagent Kit includes secondary antibodies conjugated to AzureSpectra 700 and AzureSpectra 800 to enable the simultaneous detection of two biomarkers. For best results, use with a Sapphire FL Biomolecular Imager.

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.

Products for In-cell Western Blotting

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

Reagents Needed for In-cell Westerns
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.

In-cell Western App Notes

Related Applications

Looking for a full list of applications?

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In-cell Western taken from Azure Sapphire Biomolecular Imager
In-cell Western

History Behind In-cell Westerns

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

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