In-cell Westerns are a powerful and versatile technique for studying cellular behaviors at a molecular level. By combining the specificity of a traditional Western blot with the high-throughput nature of an ELISA, in-cell Westerns let researchers capture even the most subtle variations in protein expression, provided they are properly designed.
In-cell Westerns, also known as cell-based ELISAs, cytoblots, or in-cell ELISAs, are a fusion of traditional Western blots and ELISAs. They involve plating cells into 96- or 384-well microplates and carrying out any necessary experimental treatments before fixing, permeabilizing, and blocking. Proteins of interest are then detected using target-specific primary antibodies and fluorescently labeled secondaries, prior to data capture with a suitable imaging device. Usually, the secondary antibodies are labeled with near-infrared (NIR) fluorophores to minimize interference from auto-fluorescence or noise from the microplate plastic. A nuclear stain can be used to normalize the signal to the number of cells per well. The in-cell Western workflow is shown in Figure 1.
Figure 1. The in-cell Western workflow showing detection of a single protein target.
A defining feature of in-cell Westerns is that they do not call for a protein extraction step, as required by both traditional Western blotting and ELISA. Instead, in-cell Westerns detect cellular proteins in situ, providing more physiologically relevant results in faster time and with less risk of target antigens being lost or damaged. A further important difference between in-cell Westerns and traditional Western blotting (using enzyme-labeled antibodies and chemiluminescent detection) is that in-cell Westerns have a much higher throughput. This is because they eliminate the need to perform polyacrylamide gel electrophoresis (PAGE) and protein transfer, as well as allow for quantifying up to four different targets per well.
In-cell Westerns have utility for a broad range of research applications. These include studying the effects of small molecule drugs on cell signaling pathways, investigating changes to protein expression following genetic manipulation or post-translational modification (PTM), and assessing viral titers to understand how viruses replicate in different types of host cells. In-cell Westerns are also used for monitoring protein localization within specific cellular compartments, identifying cellular changes in disease models, and evaluating protein-protein interactions.
The reagents needed to perform an in-cell Western include the fixative and permeabilization solutions (typically, 100% methanol is used for simultaneous fixation and permeabilization), blocking solution, total stain, and both primary and secondary antibodies. For convenience, all these components (except for the primary antibodies) may be purchased as a single off-the-shelf product, such as the AzureCyto In-Cell Western Kit from Azure Biosystems. This kit is available with different NIR secondary antibody options (goat anti-mouse and goat anti-rabbit) and includes the highly sensitive AzureCyto Total Cell Stain (for incubation with primary antibodies) that makes normalization at low concentrations possible. Access to a multi-channel imaging device that is fitted with suitable lasers and detectors and is capable of bottom reading microplates is also necessary. Critically, this should feature minimal crosstalk to safeguard data quality.
The Sapphire FL Biomolecular Imager is ideally suited to in-cell Westerns due to its sensitivity, speed, and high (5-micron) resolution, as well as its capacity to support multiplexed detection. The Sapphire FL is widely used for looking at total and modified (e.g., phosphorylated) versions of the same protein while also detecting an in-well standard to ensure accurate quantification across an entire plate. Figure 2 provides an example of this type of data, with the composite image demonstrating a lack of crosstalk between the signal channels.
Figure 2. 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.
Further advantages of the Sapphire FL include software that provides precise focal plane adjustment and an automated Z-scanning function that is used to find the focus of microplates of different brands. This affords a high level of sensitivity and specificity, as shown in Figure 3. In addition, the Sapphire FL is fitted with customizable and user-changeable laser and filter modules spanning the UV, visible, and NIR spectra that allow for easy adaptation to researchers’ changing needs.
To learn more about in-cell Westerns and the Sapphire FL Biomolecular Imager, visit azurebiosystems.com/in-cell-westerns
Figure 3. 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.