On-cell Westerns

What is the on-cell Western assay?

The on-cell Western (OCW) assay is a whole-cell immunodetection assay used to monitor cell surface protein expression. Like the related in-cell Western (ICW) assay, the OCW allows for high throughput, quantitative analysis of protein expression. An example of an on-cell Western is shown in Figure 1.

On-cell Western assay
Figure 1. On-cell Western

What on-cell Westerns are used for

The OCW assay is a highly versatile and useful tool that can be employed for a wide variety of purposes. Typically, a single cell surface antigen is measured. With multiplex fluorescence detection, two targets can be studied in each sample, which allows studies comparing the levels of phosphorylated and unphosphorylated forms of a protein.

Among its many applications, OCW is particularly useful for:

  • The detection and quantification of target proteins that are localized to the surface of cells,
  • Quantifying ligand binding to cell surface receptors, as well as to monitor receptor internalization and recycling by detecting loss and reappearance at the cell surface, and
  • Performing and detecting cell surface biotinylation assays, allowing researchers to evaluate the effects of mutations, targeted therapeutics, and other treatments on protein expression and trafficking.

Similarities between on-cell Westerns and in-cell Westerns

Because they are both high throughput assays, both the ICW and OCW approaches can be used as screening tools in drug discovery.

Differences between on-cell Western assay and in-cell Western

Unlike an ICW, where cells are permeabilized before probing for the target protein, an OCW assay is used exclusively to monitor cell surface protein expression (Figure 2). The OCW also differs from an ICW in that the cells are not permeabilized before adding the primary antibody.

Figure 2. Figure 5 from Fisher et al. J Biol Chem. 2012;287(26):21673-21685. Licensed under CC BY 4.0.

What information do on-cell Westerns provide?

On-cell Westerns do not provide information about antigen molecular weight.

On-cell Western benefits

By skipping the protein extraction, gel separation, and membrane transfer steps, the OCW assay saves time compared to traditional Western blotting while allowing cell surface proteins to be studied in a more biologically relevant context.

On-cell Western Protocol

The OCW assay begins by seeding cells into the wells of a flat-bottomed, clear microplate, as you would for an ICW. The cells are cultured until the desired cell density is achieved and then probed with an antibody for an antigen of interest. Similar to a traditional Western blot, the OCW involves a blocking step to prevent nonspecific antibody binding, wash steps to remove unbound antibody, and incubation with a labeled secondary antibody to detect the primary antibody.

The secondary antibody can be labeled with peroxidase and detected using a colorimetric or chemiluminescent substrate. Often the secondary antibody is labeled with a fluorophore and detected using a fluorescence imaging system.

Near-infrared fluorophores are preferred because these excitation and emission wavelengths suffer from less background autofluorescence from biological materials and plastic labware (Boveia 2015).

The cells in both on-cell and in-cell Western assays can be imaged either live or post-fixation.

Learn more about in-cell Westerns here

How to image on-cell Westerns

The imaging approach used for OCW and ICW assays depends on the label on the secondary antibody. Colorimetric substrates can be imaged using a microplate reader. Fluorescence detection requires a fluorescence imaging system that can image multi-well plates, such as 96-well plates.

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 imaging on-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 96-well plates like a pro. Learn more about the breadth of applications the Sapphire FL is capable of and schedule a free demo by clicking here.

Publications

Since its introduction, the OCW technique has since been adapted to study multiple cell surface proteins and a variety of disease states including viral infection. As of 2024, the term “on-cell western” retrieves 15 publications from PubMed and seven publications are retrieved with the terms “cell-based ELISA” plus surface expression. Some results from publications that represent the power of the approach are summarized in Table 1.

Table 1. Publications featuring on-cell Westerns

Publications
Fisher et al used OCW to assess the stability of expression of wild type and mutant CFTR (the cystic fibrosis transmembrane receptor) protein in cultured cells. As shown in Figure 2, OCW experiments contributed to their conclusion that the mutant protein has an increased half-life and increased membrane stability compared to wild type (Fisher 2012). These authors used a secondary antibody conjugated to an infrared fluorescent dye and imaged their experiments with an infrared imaging system (Fisher 2012).
Bridge et al used an on-cell assay to measure binding of photoactive antibodies to EGFR on the cancer cell surface. They demonstrate that they can used light to control delivery of small molecules to cancer cells with their engineered photoactive antibody. In their assay the secondary antibody was labeled with HRP and a plate reader was used to measure chemiluminescence (Bridge 2019).
Li et al used a cell-based ELISA on non-permeabilized cells in their work demonstrating binding of a therapeutic antibody to breast cancer cells. The authors developed a novel humanized antibody directed towards MUC1, a membrane protein that is a potential target in breast and pancreatic cancer. Their cell-based ELISA was detected on a microplate reader after using an HRP-conjugated secondary antibody and a colorimetric HRP substrate (TMB) (Li 2023).

REFERENCES

  1. Boveia, V., & Schutz-Geschwender, A. (2015). Quantitative analysis of signal transduction with in-cell western immunofluorescence assays. Detection of Blotted Proteins: Methods and Protocols, 115-130.

  2. Bridge T, Shaikh SA, Thomas P, et al. Site‐Specific Encoding of Photoactivity in Antibodies Enables Light‐Mediated Antibody–Antigen Binding on Live Cells. Agnew Chem Int Ed Engl. 2019;58(50):17986-17993.

  3. Chen H, Kovar J, Sissons S, et al. The majority of ACTH receptor (MC2R) mutations found in Familial Glucocorticoid Deficiency type 1 lead to defective trafficking of the receptor to the cell surface. Anal Biochem. 2005;338(1):136-142.

  4. Chung TT, Webb TR, Chan FL, et al The majority of ACTH receptor (MC2R) mutations found in Familial Glucocorticoid Deficiency type 1 lead to defective trafficking of the receptor to the cell surface. J Clin Endocrinol Metab. 2008;93(12):4948-4954.

  5. Egorina EM, Sovershaev MA, Bjørkøy G, et al. Intracellular and surface distribution of monocyte tissue factor: application to intersubject variability. Arterioscler Thromb Vasc Biol. 2005;25(7):1493-1498.

  6. Egorina EM, Sovershaev MA, Østerud B. In-cell Western assay: a new approach to visualize tissue factor in human monocytes. J Thromb Haemost. 2006;4(3):614-620.

  7. Fisher JT, Liu X, Yan Z, et al. Comparative processing and function of human and ferret cystic fibrosis transmembrane conductance regulator. J Biol Chem. 2012;287(26):21673-21685.

  8. Foreman KE, Vaporciyan AA, Bonish BK, et al. C5a-induced expression of P-selectin in endothelial cells. J Clin Invest. 1994;94(3):1147-1155.

  9. Gonzalez-Gronow M, Kaczowka SJ, Payne S, Wang F, Gawdi G, Pizzo SV. Plasminogen structural domains exhibit different functions when associated with cell surface GRP78 or the voltage-dependent anion channel. J Biol Chem. 2007;282(45):328111-32820.

  10. Li L, Cao J, Chen C, et al. Antitumor effect of a novel humanized MUC1 antibody-drug conjugate on triple-negative breast cancer. Heliyon. 2023;9(4):e15164.

  11. Mulligan MS, Vaporciyan AA, Miyasaka M, Tamatani T, Ward PA. Tumor necrosis factor alpha regulates in vivo intrapulmonary expression of ICAM-1. Am J Pathol. 1993;142(6):1739-1749.

  12. Stockwell BR, Haggarty SJ, Schreiber SL. High-throughput screening of small molecules in miniaturized mammalian cell-based assay involving post-translational modifications. Chem Biol. 1999;6(2):71-83.

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