How to Troubleshoot Common In-cell Western Issues

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Basic guide to mastering In-Cell Western assays

In-cell Westerns (ICW) assays are powerful and versatile tools in the arsenal of cell biologists and protein research. These assays offer the unique advantage of quantifying target proteins and assessing protein expression and activation directly within the native cellular context, providing valuable insights into cellular processes, signaling pathways, and treatment responses. 

Like any experimental technique, ICWs are not immune to challenges and pitfalls. From poor antibody specificity and high background to nuances in staining, imaging platforms and quantification, these challenges can hinder progress and data interpretation.

In-cell western assay
In-cell Western assay performed using AzureCyto In-cell Western Kit. A 96-well plate was imaged on the Azure Sapphire FL using the 532, 685, and 784 Standard Optical Modules at a resolution of 10µm with the focus set to +3.00mm.

Use this basic guide to aid you in troubleshooting the common issues encountered during in-cell Western assays. Whether you are a seasoned investigator or just beginning to explore the world of ICW, this is a resource that will equip you with the knowledge and strategies you need to successfully navigate unique intricacies.


Common issues with In-cell Westerns

Pipette Carefully

A great rule of thumb to follow when working with cells is to never touch the bottom of the
ICW plate (or any plate for that matter) with the tip of the pipette. Touching the bottom of the plate causes defects and prevent the cells from adhering properly.

  • Pipette by touching the sides of the wells.

    To ensure the cells adhere evenly, gently tap each side of the plate a few times after seeding.

Cell Quality and Health

Poor cell health can lead to unreliable results.

  • Verify your cells are healthy, adherent, and at the appropriate confluence before initiating the assay.

  • Check for signs of contamination or cell detachment.

Determine Linear Range

For quantitative results, the number of cells plated and the concentration of primary antibody both need to be optimized prior to testing. In the case below, plating 100,000 cells per well resulted in inconsistencies since the cells were too confluent and began to peel off of the surface of the plate along the edge of the wells. Maximum linearity was observed when 1563-25,000 cells/well were used. This antibody showed good linearity at all dilutions used during optimization, so a 1:200 dilution is suitable to conserve precious and expensive antibody.

Fixation and Permeabilization

Inadequate fixation and permeabilization can result in incomplete antibody penetration and uneven staining.

  • Optimize fixation and permeabilization conditions for your specific cell type.

  • Adjust fixation and permeabilization times, concentrations, or temperatures if necessary.

  • Use a proven and robust permeabilization solution, such as AzureCyto Permeabilization solution

    The AzureCyto In-Cell Western Reagent Kit contains all reagents necessary for ICWs, including a permeabilization solution. The permeabilization solution provided with the AzureCyto Kit removes membrane lipids and facilitates permeabilization of the nuclear membrane to allow the penetration of antibodies into cells for the detection of intracellular and nuclear proteins.

  • AzureCyto In-cell Western Kit with fluorescent secondary antibodies

    AzureCyto In-Cell Western Kit

    The AzureCyto In-Cell Western Kit contains all reagents necessary for staining of whole cells for total cell normalization and simultaneous detection of two biomarkers. The …
    $749.00 (USD)

Antibody specificity

Incubating antibodies with whole cells can cause non-specific binding due to increased sample complexity

  • How to validate that your antibody can be used with the ICW technique:

    Block the antibody with a 10-fold excess of immunogen (peptide or protein) for 30 minutes prior to the antibody incubation step on the plate.


Insufficient blocking can lead to high background signals.

  • Use an appropriate blocking buffer and incubate cells for an adequate duration.

    The block solution provided in the AzureCyto Kit is specially formulated for cell-based assays and fluorescence detection.

  • Test different blocking buffers and concentrations to minimize background.

Primary Antibody

Your choice of primary antibodies matters. Incubating antibodies with whole cells can cause non-specific binding due to increased sample complexity. Be careful: using low quality primary antibodies can yield weak or nonspecific signals.

How to select the best primaries to use when dealing with in-cell Westerns:

  • Select validated antibodies with high specificity for your target protein.

    To validate that your antibody can be used with the ICW technique, we suggest blocking the antibody with a 10-fold excess of immunogen (peptide or protein) for 30 minutes prior to the antibody incubation step on the plate.

  • Optimize antibody concentration and incubation time.

Secondary Antibody

Just like with primary antibodies, your choice of secondary antibodies matters too. If you use inappropriate or cross-reactive secondary antibodies, it can result in nonspecific staining.

Three tips for using appropriate secondary antibodies that provide quality results:

  • Choose secondary antibodies that are highly specific to the host species and isotype of your primary antibody.

  • Perform control experiments to validate secondary antibody specificity.

  • Choose fluorophores with excitation and emission ranges with little to no overlap.

    Azure offers secondary antibodies labeled with fluorophores, which emit light in visible and near-infrared wavelengths. The AzureSpectra 490, 550, 650, 700 and 800 secondary antibodies offer unparalleled sensitivity and performance for immunoblotting applications when used in conjunction with Azure Imaging Systems.


Variations in incubation conditions can affect staining consistency. Overnight incubation times can lead to increased background.

  • Maintain consistent incubation times, temperatures, and agitation during antibody incubations.

  • Edge effects

    It is common to seed the wells of tissue culture plates with 100-200μL/well of cells in media. If the cells require incubation for more than one day prior to ICW, feed them with 50-100μL/well of fresh media. This will ensure that the cells have sufficient media for continued growth as well as prevent the wells from drying out.

common to seed the wells of tissue culture plates
Ensure there is a sufficient level of humidity inside the incubator. This can be achieved by placing a water tray filled with distilled water at the bottom of the incubator. It is typical for the wells along the edges of the plate to be impacted the most when the cells do not have sufficient media


Inadequate washing can leave residual unbound antibodies, leading to high background.

  • Ensure thorough and consistent washing between incubation steps.

  • Use an appropriate wash buffer and follow recommended wash protocols.


Using improper imaging settings or imagers with high crosstalk can result in inaccurate data.

  • Use lasers imagers with desired fluorophores for crosstalk in the proper wavelengths.

    The Sapphire FL is capable of imaging in-cell Westerns very well, 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 like a pro.

  • Test fluorophores and antibody pairs to determine the correct imaging settings for linearity.

  • Use consistent settings for capturing images and quantifying signals.


Choosing the wrong normalization method can lead to misinterpretation of results.

  • Evaluate the suitability of normalization options based on your experiment.

  • Consider using total cell staining as a normalization method, especially for comparing protein levels across different samples, such as treated vs untreated cell lines.

  • Use a total stain with high linearity at the cell volume range of your experiment.

    The AzureCyto Total Cell Stain is a sensitive and linear stain that can be co-incubated with primary antibodies to streamline the detection process and reduce the number of assay steps. It is included in the AzureCyto In-cell Western Reagent Kit.

Replicates and Controls

Inadequate replicates or lack of appropriate controls can compromise data quality.

  • Include sufficient biological and technical replicates in your experiment.

  • Utilize positive and negative controls to validate assay performance.

1:2 serial dilution of HeLa cells seeded into a 96-well plate
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.

Using an in-cell Western kit such as the AzureCyto In-Cell Western Kit can cut down on the time spent troubleshooting and optimizing your assay. The AzureCyto In-Cell Western Kit enhances the consistency and reliability of results by providing researchers with validated reagents, standardized protocols, and quality controlled components reducing experimental variability and ensuring accurate and reproducible data.

Additional reading regarding in-cell Westerns

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